Bca protein assay kit 2

Cells were dissolved by RIPA reagent (HEART Biotech, Xi’an, China). Protein levels were quantified by BCA protein assay kit II (#5000002, BIO-RAD, Hercules, CA, USA). Lysates (40 μg protein) were separated by 10% SDS-PAGE gel and transferred onto a PVDF membrane (IPVH00010, Millipore, Billerica, MA, USA). Primary antibodies of TRAF6, Akt (#4691), Phospho-Akt^Thr308 (#13038) and β-actin (#3700) were purchased from CST (Danvers, MA, USA). Primary antibodies of Bcl-2 (12789-1-AP), Mcl-1 (16225-1-AP) and MMP-9 (10375-2-AP) were purchased from PROTEINTECH (Rosemont, IL, USA). PVDF membranes with certain proteins were incubated with matched antibodies (diluted as 1:1000) overnight. Secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (diluted as 1:5000, ABGENT, San Diego, CA, USA) were used to bind matched primary antibodies. Protein expression levels were visualized by Enhanced chemiluminescence reagent (WBKLS0500, Millipore, Billerica, MA, USA) and calculated by Image J software.

Cells lysed in RIPA buffer (Beyotime) were subjected to BCA protein assay kit II (BIO‐RAD). After separation by 10% SDS‐PAGE gel, protein samples were transferred onto nitrocellulose membranes (Invitrogen) and incubated with rabbit‐anti‐human MYC (ab9106; Abcam) and rabbit‐anti‐human GAPDH (ab9485; Abcam) antibodies overnight at 4°C. Horseradish peroxidase (HRP)‐conjugated secondary antibodies were subsequently used to incubate samples for 1 hour. The final blot signals on the protein samples were observed by ECL reagents (Millipore).

HCC tissues and cells were lysed with RIPA buffer (Beyotime, Shanghai, China) and protein concentrations were quantified with a BCA protein assay kit II (BIO-RAD, Hercules, CA, USA). Protein samples were separated by 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Invitrogen). The membranes were incubated with rabbit-anti-human FOXA1 (ab170933; Abcam, Cambridge, MA, USA), rabbit-anti-human PARP1 (ab191217; Abcam), rabbit-anti-human caspase-3 (ab32351; Abcam), rabbit-anti-human caspase-7 (ab32522; Abcam), rabbit-anti-human Cyclin D1 (ab134175; Abcam), rabbit-anti-human p21 (ab109520; Abcam), mouse-anti-human β-actin (ab8226; Abcam) and mouse-anti-human GAPDH primary antibody (sc-47,724; Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA) overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (NXA931–1ML and NA934-1ML, GE Healthcare Life Sciences, Beijing, China) were used to incubate the membranes for 1 h at room temperature. The blot signals on the membrane were visualized with ECL reagents (Millipore, Plano, TX, USA) and detected using Amersham™ Imager 680 from GE Healthcare Life Sciences.