Anti ptbp2 antibody

The Western blotting analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-PTBP1 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-PTBP2 antibody (Abcam, Cambridge, UK), anti-PTBP3 antibody (Sigma-Aldrich, St. Louis, MO, USA), anti-HNRNPK antibody (Abcam), anti-HNRNPM antibody (Sigma-Aldrich), anti-FUBP3 antibody (Abcam), anti-CPSF2 antibody (Abcam), anti-G3BP2 antibody (Atlas Antibodies), anti-TGFβ1 antibody (Proteintech Group), anti-TGFβ2 antibody (Abcam), anti-p-SMAD2 antibody (Cell Signaling Technology), anti-SMAD2 antibody (Cell Signaling Technology), anti-p-SMAD3 antibody (Cell Signaling Technology), anti-SMAD3 antibody (Cell Signaling Technology), anti-SMAD5 antibody (Abcam), anti-ID2 antibody (Abcam), anti-ZEB1 antibody (Abcam), anti-SNAI antibody (Abcam), anti-E-cadherin antibody (Proteintech Group), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology) and anti-GAPDH antibody (Sigma-Aldrich). The blots were incubated with a goat anti-rabbit or anti-mouse secondary antibody (Sigma-Aldrich) and visualized with a commercial ECL kit (Pierce, Rockford, IL).

To assess the degree of damage to the sperm cells, hematoxylin and eosin (HE) staining was carried out to evaluate testicular histological changes in the mouse model. In addition, localization of the Ptbp2 protein in the testicular tissues of mice was determined by immunohistochemistry (IHC). The testicular samples were fixed with 4% paraformaldehyde, then embedded in paraffin wax and sectioned at 4 μm. The testicular tissue sections were deparaffinized in different concentrations of dimethylbenzene and ethanol solutions, then heated in sodium citrate buffer (pH 6.0) in a microwave for 20 min. Next, the sections were dipped in deionized water containing 3% H₂O₂ to quench endogenous peroxidase activity. After specific treatment with 10% normal goat serum to block non-specific binding, the deparaffinized sections were incubated overnight at 4°C with the anti-PTBP2 antibody (Abcam) at 1:300 dilution, then incubated with a goat anti-rabbit biotinylated secondary antibody for 40 min at room temperature. The immunoreactivity with Ptbp2 was observed using streptavidin-peroxidase and 3,3N-diaminobenzidine (Beyotime, Jiangsu, China).