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SMURF2 / E3 ubiquitin-protein ligase SMURF2 · IHC design guide
Design Immunohistochemistry for SMURF2
Plan chromogenic SMURF2 IHC-P with catalog antibody A02585 at 2 μg/mL (datasheet). Use testicular spermatogonia as a high-staining reference and assess cytoplasmic staining across tissues (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SMURF2 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic in most tissues; high in spermatogonia (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Some cell types, including adipocytes, lack detectable staining (HPA tissue IHC) | |
| Regulation | SMAD7 promotes SMURF2 autodegradation (UniProt) | |
| Isoform / epitope | No isoforms reported; one 1–748 chain (UniProt) |
Section 1
Recommended SMURF2 IHC & IF Protocols
The catalog antibody protocol is paired with published SMURF2 IHC methods for breast cancer tissues (PMC3918234), human osteoarthritic cartilage (PMC2636703), and pancreatic cancer tissue microarrays (PMC12133423).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A02585); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-SMURF2, 2 μg/mL (datasheet A02585) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SMURF2-positive staining in spermatogonia cells of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA HIER at pH 9.0, 95–98 °C for 20 min (page retrieval: nuclear antigen); compare the published retrieval where specified (PMC3918234).
Section 2
What Is the Expected SMURF2 Staining Pattern?
In paraffin-section IHC, expect predominantly cytoplasmic SMURF2 staining across many tissues, with high staining in testicular spermatogonia and medium staining in several other listed cell populations (HPA: tissue IHC). UniProt also places SMURF2 in the nucleus and at the cell membrane or membrane rafts, despite reporting no transmembrane segment (UniProt Q9HAU4: subcellular location and topology). The HPA tissue-IHC assessment is Enhanced, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC).
| Cytoplasmic staining is strongest in spermatogonia; adrenal glandular cells, Purkinje cells, and kidney tubular cells show more moderate staining. | This fits the reported high spermatogonial and medium staining in those other cell populations (HPA: tissue IHC). Compare compartments and cell identities within each section; the listed levels describe observations, not a required intensity ratio between separately processed slides (HPA: tissue IHC; general IHC practice). |
| Staining is exclusively nuclear, or a sharp membrane rim dominates nearly every cell, with little of the expected cytoplasmic pattern. | Check whether the pattern is reproducible and antibody dependent before calling it SMURF2: HPA describes predominantly cytoplasmic tissue staining, while UniProt also lists nuclear and membrane locations (HPA: tissue IHC; UniProt Q9HAU4: subcellular location). Nuclear signal alone is therefore not proof of an artefact; a widespread membrane rim should not be treated as evidence of a transmembrane protein (UniProt Q9HAU4: topology). |
| Strong staining appears in adipocytes, cervical squamous epithelium, or lymph-node germinal-center cells. | These specific cell populations were reported as not detected, making a strong result discordant with the HPA reference pattern (HPA: tissue IHC). Investigate antibody cross-reactivity or endogenous chromogen-generating activity with appropriate controls before assigning the signal to SMURF2 (general IHC practice). |
| Color coats extracellular spaces, section edges, or many unrelated cell populations without a defined intracellular pattern. | Treat this as background until controls show otherwise: nonspecific binding, insufficient washing, and endogenous detection activity can obscure a cellular chromogenic signal (general IHC practice). The reference pattern is cytoplasmic expression in most tissues, not diffuse staining throughout the section (HPA: tissue IHC). |
| Spermatogonia are unstained in a section selected as a positive control. | A negative result conflicts with the reported high spermatogonial staining (HPA: tissue IHC). Check that the expected cells are present, then assess retrieval, antibody and detection performance with controls (general IHC practice). A failed run cannot establish that this specimen lacks SMURF2. |
💡Expected SMURF2 appearanceCall a result positive when identifiable spermatogonia show strong, mainly cytoplasmic chromogenic staining, with moderate staining possible in the listed positive populations; widespread extracellular color or strong staining in HPA-listed undetected cells warrants control checks (HPA: tissue IHC; general IHC practice).
| Tissue and cell selection | SMURF2 is widely expressed with low RNA tissue specificity, but staining differs by cell population: spermatogonia are high, several listed populations are medium, and selected populations are undetected (UniProt Q9HAU4: tissue specificity; HPA: tissue IHC). Name the cells being scored rather than treating an entire organ as uniformly positive or negative. |
| Compartment and protein topology | HPA reports mostly cytoplasmic tissue staining; UniProt also lists nucleus, cell membrane, and membrane rafts, with cytoplasmic localization in the presence of SMAD7 (HPA: tissue IHC; UniProt Q9HAU4: subcellular location). SMURF2 has no transmembrane segment, so a membrane-associated signal does not imply membrane-spanning topology (UniProt Q9HAU4: topology). |
| Antibody evidence and its limit | HPA lists antibody HPA071508 as Enhanced for IHC, while its tissue summary reports medium agreement between antibody staining and RNA expression (HPA: antibody validation; HPA: tissue IHC). Use that evidence to anchor expected patterns, while checking discordant cell populations with run controls (general IHC practice). |
| IF/ICC Q: What pattern should I expect? | A: HPA reports supported localization to nuclear speckles in ICC-IF images from BJ, U-251MG, and U2OS cells (HPA: subcellular ICC-IF). This observation comes from cultured-cell fluorescence imaging; interpret paraffin-section chromogenic staining against the tissue-IHC pattern instead (HPA: subcellular ICC-IF; HPA: tissue IHC). |
| Epitope and retrieval evidence | UniProt lists an intact 748-aa chain, no signal peptide or propeptide, and no listed glycosylation sites; the supplied evidence does not map the IHC antibody epitope or establish SMURF2-specific retrieval or fixation sensitivity (UniProt Q9HAU4: processing and glycosylation; HPA: antibody validation). Treat retrieval optimization as a general IHC workflow decision, not a predicted SMURF2 effect (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in the expected positive cells | The run may have inadequate antigen exposure, inactive detection reagents, or missing target cells; these are general IHC possibilities, not established SMURF2-specific fixation effects (general IHC practice). | Confirm spermatogonia are present in the control section and assess the antibody, retrieval, and detection steps with run controls (HPA: High in spermatogonia; general IHC practice). |
| Strong staining in a reference undetected population | A strong signal in adipocytes or cervical squamous cells conflicts with the HPA observations; antibody cross-reactivity or endogenous detection activity are possibilities (HPA: Not detected in those cells; general IHC practice). | Compare a no-primary control and a known-positive section, then check endogenous enzyme blocking for chromogenic detection before scoring the signal as SMURF2 (general IHC practice). |
| Diffuse color obscures cell boundaries | Nonspecific reagent binding, incomplete washing, or detection background can reduce contrast (general IHC practice). | Check the no-primary control; review blocking, washes, antibody working concentration, and chromogen development while retaining a positive control (general IHC practice). |
| A nuclear pattern conflicts with the expected tissue-IHC image | Nuclear localization is biologically plausible, but HPA's paraffin-tissue summary is predominantly cytoplasmic; a nuclear-only result needs verification (UniProt Q9HAU4: subcellular location; HPA: tissue IHC). | Compare matched positive and no-primary controls, confirm cell identity, and evaluate reproducibility before interpreting nuclear signal as SMURF2 (general IHC practice). |
| Membrane-edge color dominates the section | UniProt permits membrane-raft association, but SMURF2 has no transmembrane segment; uniform edge staining may also reflect technical background (UniProt Q9HAU4: subcellular location and topology; general IHC practice). | Check whether staining follows specific cells rather than section edges; compare controls and the expected cytoplasmic tissue pattern (HPA: tissue IHC; general IHC practice). |
| IF/ICC nuclear speckles and tissue-IHC cytoplasmic staining seem inconsistent | The observations come from different assay contexts: HPA supports nuclear speckles in cultured-cell ICC-IF and describes cytoplasmic expression in most tissue-IHC samples (HPA: subcellular ICC-IF; HPA: tissue IHC). | Record assay, specimen, and compartment separately; score the paraffin section against its tissue-IHC reference and assess the IF/ICC image against its own subcellular reference (HPA: tissue IHC; HPA: subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing SMURF2 staining
A02585 · IHCImmunohistochemistry of SMURF2 in mouse liver tissue with SMURF2 antibody at 5 μg/mL.
A02585 · IHCImmunohistochemistry of SMURF2 in human uterus tissue with SMURF2 antibody at 2 μg/ml.
A02585 · IFImmunofluorescence of SMURF2 in mouse liver tissue with SMURF2 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000108854-SMURF2/tissue/cerebral+cortex#imid_19745683
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000108854-SMURF2/tissue/stomach+1#imid_19742991
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000108854-SMURF2/tissue/colon#imid_19743025
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000108854-SMURF2/tissue/liver#imid_19743026
![HPA ICC-IF BJ [Human fibroblast] — SMURF2 (HPA071508)](https://images.proteinatlas.org/71508/1533_A8_1_blue_red_green.jpg)
HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA071508; green = SMURF2, blue = nucleus, red = microtubules.
![HPA ICC-IF BJ [Human fibroblast] — SMURF2 (HPA071508)](https://images.proteinatlas.org/71508/1533_A8_2_blue_red_green.jpg)
HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA071508; green = SMURF2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SMURF2 IHC & IF
🧪Run testis first and expect staining in spermatogonia (HPA: High in spermatogonia). Use adipose tissue as a negative comparison, focusing on adipocytes (HPA: Not detected in adipocytes); on the testis slide, compare spermatogonia with neighboring cells that show only background staining, without assuming all other testicular cells are negative.
Positive control tissue: Testis (Spermatogonia cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SMURF2 in BJ [Human fibroblast], U-251MG, U2OS, with annotated localisation: Nuclear speckles (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host-species- and isotype-matched control antibody for a monoclonal primary, or matched nonimmune IgG for a polyclonal primary; knockout tissue, if available, provides a biological negative. For chromogenic IHC in testis, check and block endogenous peroxidase; for IF, inspect an unstained section for autofluorescence.
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval against the positive and negative tissue controls. The selected A02585 mouse-liver IHC caption reports 5 µg/mL but does not state a fixative (caption: fixative unreported). The evidence does not establish that frozen sections or IF are easier, or identify a SMURF2-specific testis artefact; nuclear speckles are a supported ICC-IF location to assess if IF is performed (HPA: Nuclear speckles supported).
HPA tissue IHC evidence for SMURF2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Testis | Spermatogonia cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SMURF2 IHC Tips
Troubleshoot SMURF2 staining in paraffin sections by checking retrieval, cellular compartment, controls and scoring before interpreting chromogenic signal.
What retrieval conditions should I use when SMURF2 staining is weak?
Start with Tris-EDTA, pH 9.0, heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval setting). If staining remains weak, compare a shorter exposure or a citrate buffer at pH 6.0 on adjacent sections while holding antibody concentration and detection constant; these are fallback optimization conditions, not reported SMURF2 results (standard IHC practice). Allow sections to cool consistently, then assess staining against a positive control and a no-primary control (standard IHC practice). Record nuclear and cytoplasmic signal separately because SMURF2 can occupy both compartments (UniProt Q9HAU4 subcellular location).
Could fixation explain weak or uneven SMURF2 staining?
Target-specific fixation sensitivity is unknown from the supplied evidence; the selected mouse liver image states neither its fixative nor its fixation time (A02585 tissue-IHC caption). For paraffin sections, compare specimens with documented fixation histories and keep section thickness, retrieval and detection conditions matched (standard IHC practice). If only one batch stains weakly, repeat staining with a control section in the same run before changing antibody concentration (standard IHC practice). Record tissue preservation and staining near section edges, since uneven processing can complicate interpretation of any chromogenic IHC result (standard IHC practice).
Should I expect nuclear or cytoplasmic SMURF2 staining?
Score nuclear and cytoplasmic staining separately: SMURF2 is reported in both compartments, as well as at the cell membrane and in membrane rafts (UniProt Q9HAU4 subcellular location). Cytoplasmic localization can occur in the presence of SMAD7, while membrane raft colocalization with CAV1, SMAD7 and the TGF-beta receptor is reported (UniProt Q9HAU4 subcellular location). Tissue IHC most often shows cytoplasmic expression, whereas cell-based IF supports nuclear speckles (HPA tissue IHC profile; HPA subcellular summary). A diffuse rim in every cell, especially alongside tissue edges, warrants a no-primary control and compartment-specific review before being called membrane-associated SMURF2 (standard IHC practice).
How should epitope uncertainty affect antibody selection and staining interpretation?
The supplied record lists 0 isoforms and a 1–748 chain, but it does not identify the catalog antibody's epitope (UniProt Q9HAU4 isoforms and processing; A02585 tissue-IHC caption). SMURF2 contains a C2 domain, 3 WW domains and a HECT domain, so domain-specific epitope access cannot be inferred without antibody mapping (UniProt Q9HAU4 domains). It has no annotated glycosylation sites or modified residues in this record, which does not establish uniform accessibility after tissue processing (UniProt Q9HAU4 annotations; standard IHC practice). Compare the staining pattern with an independently validated antibody, if available, and retain no-primary and tissue controls when testing retrieval changes (standard IHC practice).
How can IF help check the cell type and compartment seen by IHC?
Use IF as a complementary check to chromogenic IHC, and multiplex SMURF2 with a validated marker for the cell type under study; spermatogonia are a high-staining tissue example (HPA tissue IHC: High in spermatogonia cells). Choose spectrally separated fluorophores after inspecting unstained tissue for autofluorescence, and include single-label controls for bleed-through (standard IF practice). Permeabilize sufficiently to access intracellular nuclear and cytoplasmic epitopes, then compare permeabilized and minimally permeabilized controls if membrane-associated signal is being evaluated (UniProt Q9HAU4 subcellular location; standard IF practice). Nuclear speckles are supported in cell-based IF, but their visibility in a paraffin tissue section should be assessed directly rather than assumed (HPA subcellular summary; standard IF practice).
What controls help distinguish SMURF2 signal from chromogenic background?
Run a no-primary section to reveal secondary-reagent and detection background, and inspect a matched unstained section for endogenous pigment (standard IHC practice). For peroxidase-based chromogenic detection, block endogenous peroxidase before applying the detection reagents; DAB and that block are general workflow choices, not SMURF2-specific evidence (standard IHC practice). Titrate the IHC-validated antibody and detection exposure against tissue controls, keeping retrieval and counterstaining fixed during each comparison (standard IHC practice). HPA reports medium consistency between antibody staining and RNA expression, so diffuse staining alone deserves cell-level review rather than automatic acceptance as target signal (HPA reliability description).
How should I score SMURF2 staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear or cytoplasmic compartment before scoring, since SMURF2 occupies multiple locations (UniProt Q9HAU4 subcellular location). Report the percentage of positive cells and an H-score from intensity grades 0–3, or use positive-cell density per mm² when cell abundance varies substantially (standard IHC practice). Normalize positive counts to the number of eligible cells, and compare intensity only across slides stained and imaged under matched conditions (standard IHC practice). Keep compartment scores separate and document exclusion of folds, necrosis and section edges so differences in tissue quality do not drive the result (standard IHC practice).
When is an apparent SMURF2-positive region likely to be artefactual?
A convincing result follows a plausible compartment and cell pattern: tissue IHC is predominantly cytoplasmic, while nuclear and membrane-associated localization are also reported (HPA tissue IHC profile; UniProt Q9HAU4 subcellular location). Check cell identity before treating liver staining as cholangiocyte signal, because cholangiocytes were not detected in the supplied tissue profile (HPA tissue IHC: Not detected in cholangiocytes). Staining confined to edges, folds or necrotic areas should prompt review of a better-preserved region and matched controls (standard IHC practice). If signal persists without primary antibody or tracks endogenous peroxidase activity, troubleshoot the detection workflow before assigning it to SMURF2 (standard IHC practice).
Boster reagents
Best SMURF2 / E3 ubiquitin-protein ligase SMURF2 IHC Antibodies
A02585 has IHC and IF images from mouse liver and human uterus (catalog: image alts); listed reactivity includes human, mouse and rat (catalog: reactivity).
Real IHC data
Anti-SMURF2 Antibody
Cat # A02585
A02585 will render with its mouse liver IHC figure at 5 μg/mL (card: image alt). Additional IHC and IF images show mouse liver and human uterus (catalog: IHC and IF image alts).
Which to pick: For paraffin tissue IHC, choose A02585 because IHC-P is listed (catalog: applications); its own IHC captions show mouse liver and human uterus, but do not report the fixative (catalog: IHC image alts). For IF, A02585 has tissue images at 20 μg/mL (catalog: IF image alts); ICC validation is unreported (catalog: applications and image alts). For cross-species work, A02585 lists human, mouse and rat reactivity (catalog: reactivity), though its IHC images cover human and mouse only (catalog: IHC image alts); it is rabbit hosted, with clonality unreported (catalog: host and clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9HAU4 (SMUF2_HUMAN, E3 ubiquitin-protein ligase SMURF2).
- Human Protein Atlas. SMURF2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. SMURF2 subcellular location (ICC-IF): Localized to the nuclear speckles..
- Human Protein Atlas. SMURF2 antibody validation summary (1 antibodies).
- Downregulation of Smurf2, a tumor-suppressive ubiquitin ligase, in triple-negative breast cancers: involvement of the RB-microRNA axis. BMC cancer 2014 — PMC3918234.
- Induction of an osteoarthritis-like phenotype and degradation of phosphorylated Smad3 by Smurf2 in transgenic mice. Arthritis and rheumatism 2008 — PMC2636703.
- SMURF1 Downregulation Highlights Its Potential Role in Breast Cancer. International journal of molecular sciences 2026 — PMC12941280.
- SMURF2 Facilitates GAP17 Isoform 1 Membrane Displacement to Promote Mutant p53-KRAS Oncogenic Synergy. Molecular cancer research : MCR 2025 — PMC12133423.
- PubMed PMID:11163210 — UniProt-cited evidence.
- PubMed PMID:11016919 — UniProt-cited evidence.
- PubMed PMID:11158580 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SMURF2 (E3 ubiquitin-protein ligase SMURF2). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A02585 at 2 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Spermatogonia cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A02585); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-SMURF2, 2 μg/mL (datasheet A02585); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Some cell types, including adipocytes, lack detectable staining (HPA tissue IHC); Isoform / epitope: No isoforms reported; one 1–748 chain (UniProt); Regulation: SMAD7 promotes SMURF2 autodegradation (UniProt). Evidence: UniProt Q9HAU4, HPA tissue IHC · Enhanced, HPA ICC-IF · nuclear speckles., Datasheet A02585, PMC3918234, PMC2636703. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SMURF2/immunohistochemistry-ihc-guide.