Anti-SMAD2 Antibody Picoband®

Western blot analysis of SMAD2 using anti-SMAD2 antibody (A00090-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human placenta tissue lysates
Lane 2: rat liver tissue lysates
Lane 3: mouse testicular tissue lysates
Lane 4: mouse heart tissue lysates
Lane 5: mouse lung tissue lysates
Lane 6: mouse lung tissue lysates
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-SMAD2 antigen affinity purified polyclonal antibody (Catalog # A00090-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for SMAD2 at approximately 60KD. The expected band size for SMAD2 is at 52KD.

SRPX2 is elevated in fibroblasts in a TGF-β/SMADs manner. A : Western blot analysis of SRPX2 expression in HPFs following different dose of TGF-β1 induction for 24 h. B : Results for time-course Western blot analysis of SRPX2 expression in HPFs following TGF-β1 (10 ng/ml). C : Results for co-immunostaining of SRPX2 and p-SMAD2/3 in HPFs following TGF-β1 induction for 1h (up), lung sections of pulmonary fibrosis mice (middle), and lung sections from IPF patients (down). The nuclei were stained blue by DAPI, and the images were taken under original magnification ×400. D-E : Western blot (D) and RT-PCR (E) analysis of SRPX2 expression in HPFs pre-treated with SB431542 treatment following TGF-β1 induction. F-G : Western blot (F) and RT-PCR (G) analysis of SRPX2 expression in HPFs pre-treated with SIS3-HCL following TGF-β1 induction. The data are represented as the mean ± SEM of three independent experiments. ***, p < 0.001.
Index in PubMed under a CC BY license. PMID: 34093874

SRPX2 regulated TGF-β/SMADs signaling pathways by AP1 and SMAD7. A: Results for Western blot analysis of p-SMAD2, SMAD2, p-SMAD3 and SMAD3 expression in HPFs following TGF-β1 stimulation. B-C : Western blot (B) and RT-PCR (C) analysis of SMAD7 expression in HPFs following TGF-β1 induction. D : Expression of AP1 in HPFs after TGF-β1 stimulation. E : Western blot results for analysis of the levels of P-SMAD2, P-SMAD3 and SMAD7 in HPFs pre-treated with T-5224 (an inhibitor for AP-1) treatment following TGF-β1 induction. The data are represented as the mean ± SEM of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Index in PubMed under a CC BY license. PMID: 34093874

Srpx2 promoted FMT in BLM-induced pulmonary fibrosis. A : Western blot analysis of Fibronectin, Col1a1, α-SMA and Srpx2 expression in mice after BLM induction with Scrambled or Srpx2 siRNA-loaded liposomes. B : Representative images of immunostaining of Fibronectin, Col1a1 and α-SMA in the mice lung sections. The nuclei were stained blue by DAPI, and the images were taken under original magnification ×400. C : Western blot analysis of p-Smad2, p-Smad3 and Smad7 expression in mice after BLM induction. D : RT-PCR analysis of AP-1 expression in mice in each group. Six mice were included in each study group. The data are represented as the mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Index in PubMed under a CC BY license. PMID: 34093874

A diagram for mechanisms underlying SRPX2 regulation of pulmonary fibrosis. Specifically, TGF-β enhanced SRPX2 expression in a TGFβR1 and SMAD3-dependent manner. Subsequently, SRPX2 inhibited the expression of AP-1, by which it blunt SMAD7 expression and accelerate the phosphorylation of SMAD2/3. The activated TGF-β/SMADs signaling would promote the FMT and pulmonary fibrosis.
Index in PubMed under a CC BY license. PMID: 34093874

IHC analysis of SMAD2 using anti-SMAD2 antibody (A00090-1).
SMAD2 was detected in paraffin-embedded section of human colon cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-SMAD2 Antibody (A00090-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of SMAD2 using anti-SMAD2 antibody (A00090-1).
SMAD2 was detected in paraffin-embedded section of human placenta tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-SMAD2 Antibody (A00090-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of SMAD2 using anti-SMAD2 antibody (A00090-1).
SMAD2 was detected in paraffin-embedded section of mouse brain tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-SMAD2 Antibody (A00090-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of SMAD2 using anti-SMAD2 antibody (A00090-1).
SMAD2 was detected in paraffin-embedded section of rat brain tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-SMAD2 Antibody (A00090-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IF analysis of SMAD2 using anti-SMAD2 antibody (A00090-1).
SMAD2 was detected in immunocytochemical section of HELA cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL rabbit anti-SMAD2 Antibody (A00090-1) overnight at 4°C. DyLight®594 Conjugated Goat Anti-Rabbit IgG (BA1142) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of K562 cells using anti-SMAD2 antibody (A00090-1).
Overlay histogram showing K562 cells stained with A00090-1 (Blue line).To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-SMAD2 Antibody (A00090-1, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.