Ab32444

Immunohistochemistry (IHC) was performed with a two-step detection kit (Zsbio PV73 9000, China). The paraffin-embedded tissue sections were dewaxed in xylene, rehydrated in a graded alcohol system, and boiled in high-pressure autoclaved citric acid buffer (pH 6.0) for 15 min, and the peroxidase activity was quenched with 3% hydrogen peroxide for 20 min to avoid non-specific staining. The sections were washed three times with PBS followed by incubation overnight with anti-METTL8 antibody (Abcam, ab177201 at 1/200 dilution), PCNA antibody (Abcam, ab265609 at 1/200 dilution), C-myc antibody (Abcam, ab39688 at 1/100 dilution), or Cdc25C antibody (Abcam, ab32444 at 1/200 dilution) at 4°C. After that step, the sections were washed with PBS three times and incubated at room temperature for approximately 20 min with a reaction enhancer kit. This step was followed by three washes in PBS, incubation with secondary antibody at room temperature for 20 min, and staining with 3,3-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China). The sections were dehydrated and sealed after redyeing with hematoxylin.

Total protein was harvested from cells using RIPA lysis buffer (Beyotime, China). Protein samples were separated by sodium dodecyl-sulfate polyacrylamide (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membranes. Blots were washed and incubated with antibodies. The protein bands were then visualized with an ECL detection system. The primary and secondary antibodies utilized in this study were listed as follows: anti-GAPDH (60004-1-Ig, Proteintech, USA), anti-HNRNPAB Abcam, USA), anti-CDK1 (ab133327, Abcam, USA), anti-Cyclin B1 (ab32053, Abcam, USA), anti-CDC25A (sc-7389, Santa Cruz, USA), anti-CDC25C (ab32444, Abcam, USA), anti-ER (ab75635, Abcam, USA), anti-HER2 (ab134182, Abcam, USA), HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, USA), and HRP-conjugated goat anti-rabbit IgG (SA00001-2, Proteintech, USA).