Anti-YTHDF2 Antibody Picoband®

Western blot analysis of YTHDF2 using anti-YTHDF2 antibody (A02621-1).
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 K562 whole cell lysates,
Lane 2: human Raji whole cell lysates,
Lane 3: human HL-60 whole cell lysates,
Lane 4: human Hela whole cell lysates,
Lane 5: human A549 whole cell lysates,
Lane 6: human Hepg2 whole cell lysates,
Lane 7: human THP-1 whole cell lysates,
Lane 8: rat liver tissue lysates,
Lane 9: mouse testis 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-YTHDF2 antigen affinity purified polyclonal antibody (Catalog # A02621-1) at 0.25 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 1:5000 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 YTHDF2 at approximately 65KD. The expected band size for YTHDF2 is at 65KD.

YTHDF2 overexpression promotes cell proliferation and invasion in LUSC. A and B Representative immunoblot showed that the protein level of YTHDF2 was steadily up-regulated in two LUSC cell lines studied. The CCK8 assay was used to assess cell viability in NCI-H226 and SK-MES-1 cells. C and D The transwell assay and the wound-healing assay were used to assess the invasion potential and migration ability of NCI-H226 and SK-MES-1 cells. E and F Tumor size was measured twice a week. After 5 weeks, we dissected tumors from nude mice which had been injected with the indicated stable cell, then measured the tumor size and weight of nude mice injected with the indicated stable cells. G and H Immunohistochemistry showed the expression level of YTHDF2 from tumors of nude mice injected with the indicated stable cells. Data are represented by the mean ± SD of three independent experiments. *P < 0.05 vs. the vector group
Index in PubMed under a CC BY license. PMID: 34996459

YTHDF2 overexpression upregulates the protein levels of P-AKT and P-mTOR and induces EMT in LUSC cells. A The western blot detected the expression of mTOR/AKT-related proteins in NCI-H226 and SK-MES-1 cells. B The western blot detected the expression of EMT-related proteins in NCI-H226 and SK-MES-1 cells. C Using the Pearson correlation statistics, we examine the pairwise gene correlation analysis between YTHDF2 and mTOR in LUSC through GEPIA’ TCGA and GTEx expression data. D and E and F YTHDF2 overexpression resulted in significant promotion of cellular proliferation, invasion and migration in NCI-H226 and SK-MES-1 cells, while AKT Kinase inhibitor eliminated the promotion of YTHDF2 overexpression. Data are represented by the mean ± SD of three independent experiments. *P < 0.05 vs. the vector group
Index in PubMed under a CC BY license. PMID: 34996459

YTHDF2 knockdown inhibits cell proliferation and invasion in LUSC. A The western blot analyzed the expression of mTOR/AKT-related proteins in NCI-H226 and SK-MES-1 cells. B and C NCI-H226 and SK-MES-1 had YTHDF2 knocked out, resulting in significant inhibition of cell proliferation, migration and invasion. D YTHDF2 knockdown significantly inhibits cell migration viability in NCI-H226 and SK-MES-1 cells by the wound-healing assay. Data are represented by the mean ± SD of three independent experiments. *P < 0.05 vs. the vector group
Index in PubMed under a CC BY license. PMID: 34996459

YTHDF2, as a tumor promoter, may lead to a poor prognosis for LUSC patients. A Representative immunoblot showed YTHDF2 overexpression to promote METTL14 upregulation in NCI-H226, not of METTL3. Data are represented by the mean ± SD of three independent experiments. *P < 0.05 vs. the vector group. B and C Using the Pearson correlation statistics, we examine the pairwise gene correlation analysis between YTHDF2 and METTL14, YTHDF2 and METTL3 by TCGA and GTEx expression data of GEPIA. D By a log-rank test for the overall survival (OS) and disease-free survival (DFS) analysis in LUSC, we respectively investigate gene YTHDF2, METTL14, and Snail by the ‘Survival’ tab of GEPIA. E In the immunohistochemical staining results, the overall survival of LUSC patients with pN, pTNM, and YTHDF2 expression was analyzed by A Kaplan–Meier analysis
Index in PubMed under a CC BY license. PMID: 34996459

Hypoxia specifically induces YTHDF2 overexpression to activate the mTOR/AKT axis in LUSC cells. A RT-qPCR was used to investigate the alterations of mRNA levels of YTHDF2 when LUSC cells were exposed to 1% O2 for 6 h, 16 h, 24 h, 48 h, respectively. B NCI-H226 and SK-MES-1 cells were exposed with 20% O2 or 1% O2 (1% O2, 5%CO2, 94% N2) for 24 h. The protein levels of HIF-1α, YTHDF2, and P-AKT (be phosphorylated at serine 473) were analyzed by western blot. C Using the Pearson correlation statistics, we examine the pairwise gene correlation analysis between HIF-1α and YTHDF2 by TCGA and GTEx expression data of GEPIA. D The cellular growth was analyzed by CCK8 assay at different time points. E The cellular invasion and migration growths were analyzed by transwell assay. F The migration viability was analyzed by wound-healing assay. Data are represented by the mean ± SD of three independent experiments. *P < 0.05 vs. the vector group
Index in PubMed under a CC BY license. PMID: 34996459

IHC analysis of YTHDF2 using anti-YTHDF2 antibody (A02621-1).
YTHDF2 was detected in paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-YTHDF2 Antibody (A02621-1) 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 YTHDF2 using anti-YTHDF2 antibody (A02621-1).
YTHDF2 was detected in paraffin-embedded section of mouse intestine tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-YTHDF2 Antibody (A02621-1) 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 YTHDF2 using anti-YTHDF2 antibody (A02621-1).
YTHDF2 was detected in paraffin-embedded section of rat intestine tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-YTHDF2 Antibody (A02621-1) 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 YTHDF2 using anti-YTHDF2 antibody (A02621-1).
YTHDF2 was detected in immunocytochemical section of Hep 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-YTHDF2 Antibody (A02621-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) 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 RAW264.7 cells using anti-YTHDF2 antibody (A02621-1).
Overlay histogram showing RAW264.7 cells stained with A02621-1 (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-YTHDF2 Antibody (A02621-1, 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.

Flow Cytometry analysis of RH-35 cells using anti-YTHDF2 antibody (A02621-1).
Overlay histogram showing RH-35 cells stained with A02621-1 (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-YTHDF2 Antibody (A02621-1, 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.

Flow Cytometry analysis of THP-1 cells using anti-YTHDF2 antibody (A02621-1).
Overlay histogram showing THP-1 cells stained with A02621-1 (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-YTHDF2 Antibody (A02621-1, 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.