Western blot analysis was performed according to a previously established procedure
[24] (link) Seventy-two hours after transfection, cells were washed twice with PBS and lysed in RIPA protein lysis buffer with 1% protease inhibitors to obtain protein sample. Protein concentration determination was performed using a BCA protein concentration assay kit (Biosharp, Hefei, China). Protein samples were separated by 10% SDS-PAGE, and then transferred to a PVDF membrane (BioRad). The membrane was blocked and immunoblotted with anti-EZHI antibody (bs-6528R; Bioss, Beijing, China) and anti-β-actin antibody (Bioss). The gray values of protein bands were analysed using ImageJ software, and the target protein bands were normalized to β -actin which was used as the loading control. Each experiment was performed with three biological replicates.
[24] (link) Seventy-two hours after transfection, cells were washed twice with PBS and lysed in RIPA protein lysis buffer with 1% protease inhibitors to obtain protein sample. Protein concentration determination was performed using a BCA protein concentration assay kit (Biosharp, Hefei, China). Protein samples were separated by 10% SDS-PAGE, and then transferred to a PVDF membrane (BioRad). The membrane was blocked and immunoblotted with anti-EZHI antibody (bs-6528R; Bioss, Beijing, China) and anti-β-actin antibody (Bioss). The gray values of protein bands were analysed using ImageJ software, and the target protein bands were normalized to β -actin which was used as the loading control. Each experiment was performed with three biological replicates.
