Protein Extraction and Western Blot Analysis

Total proteins were firstly extracted from cells, which were washed by cold PBS and treated with RIPA lysis buffer (KeyGEN, KGP703) (supplemented with protease inhibitors 1 mmol/L phenylmethylsulfonylfluoride, 10 mg/L pepstatin, 10 mg/L aprotinin and 5 mg/L leupeptin). Protein concentrations were determined using the BCA protein assay regents (#23225, Thermo Pierce, Rockford, IL, USA). The procedure of western blot was conducted as previously described (9 (link)). Primary antibodies used in the study include anti-AKAP13 (Immunoway, YT0161; Abcam, ab99377), anti-HEG1 (Biorbyt, orb157480), anti-caspase 3 (Immunoway, YT6113), anti-caspase 3 p17 (Immunoway, YT6161). Anti-β-tubulin (Abcam, ab210797) was used as internal standard. Detection was achieved in Odyssey CLX Two-color infrared laser imaging system (LI-COR Biosciences, Nebraska, USA). Densitometric analysis of the bands was performed using ImageJ software.