Ab125727

Proteins were extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer
(Beyotime), resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and
transferred to polyvinylidene difluoride membrane. Membranes were incubated with antibody
against HOXB8 (ab125727; Abcam, Cambridge, Massachusetts) or glyceraldehyde-3-phosphate
dehydrogenase (GAPDH, ab181602; Abcam) for overnight at 4°C after blocking with fat-free
milk. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary
goat antirabbit secondary antibody (ab6721; Abcam). Band was developed using BeyoECL kit
(Beyotime) and then analyzed with Image J version 1.42 software (NI, Bethesda,
Maryland).

Total protein isolated from cultured cells using Radio Immunoprecipitation Assay (RIPA) lysis buffer and protease inhibitor (Beyotime, Haimen, Jiangsu, China) was separated at 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the protein sample was transfected to poly vinylidene fluoride (PVDF) membrane and blocked with fat-free milk, after which the primary antibodies (anti-HOXB8: ab125727, anti-Bcl-2: ab185002, anti-Bax: ab32503, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH): ab181602; Abcam, Cambridge, Massachusetts) were incubated with the membranes at 4°C for overnight. After washed with Tris-HCl buffer solution Tween (TBST), membranes were incubated with horseradish peroxidase–conjugated secondary antibody (ab6721; Abcam) at room temperature for 4 hours. Band intensity was analyzed using ImageJ software (Bethesda, Maryland) after visualized using BeyoECL kit (Beyotime).