Hrp goat anti mouse igg h l

Western blotting assays were used to identify the different expressions of protein. Modified RIPA lysis buffer was used to collect the total cell lysates. BCA Protein Assay Kit (Beyotime, Jiangsu, China) was used to standardize the concentration of different samples. Proteins were separated by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane by using Mini-PROTEAN^® Tetra (Bio-Rad, Hercules, CA, USA), and probed with primary antibodies respectively. The primary antibodies were as follows: YAP (D8H1X) XP^® Rabbit mAb (1:1000, 14074S, Cell signaling technology), SOX2 Polyclonal antibody (1:1000, 11064-1-AP; Proteintech), OCT4 Polyclonal antibody (1:1000, 11263-1-AP; Proteintech), NANOG Polyclonal antibody (1:1000, 14295-1-AP; Proteintech), CD44 Polyclonal antibody (1:2000, 15675-1-AP; Proteintech), CD133 Polyclonal antibody (1:2000, 18470-1-AP; Proteintech), GAPDH Monoclonal antibody (1:50000, 60004-1-lg; Proteintech), Alpha Tubulin Polyclonal antibody (1:2000, 11224-1-AP; Proteintech). Subsequently the membranes were incubated with corresponding secondary antibodies: HRP-Goat anti-rabbit IgG (H+L) (RS0002; Immunoway); HRP-Goat anti-mouse IgG (H+L) (RS0001; Immunoway). Blots were detected using an enhanced chemiluminescence system (S6, manufactured by CLiNX, Shanghai, China).

HepG2, SMMC-7721 cells and collected tissues were assayed using RIPA (radio-immunoprecipitation assay) buffer. BCA was used to measure total protein concentration. Proteins were then separated by 10% SDS-PAGE and transferred to PVDF (provided by Bio-Rad, Hercules, USA) membranes. The membranes were blocked with 5% nonfat milk and incubated with mono-antibodies against POLA2 (Absin, abs138967, 70KDa), GAPDH (Proteintech, 1E6D9, 36 KDa) and PD-L1 (Huabio, ET1701-41, 33 KDa). Afterward, the membranes were washed with TBST and incubated with secondary antibodies HRP* Goat Anti Mouse IgG (H + L) ( Immunoway, RS0001, China) or HRP* Goat Anti-Rabbit IgG (H + L) (RS0002, Immunoway, China). The results were measured with an enhanced chemiluminescence system. The imageQuant LAS500 was provided by GE Health Care (Fairfield, America). Chemiluminescent HRP Substrate was obtained from Millipore Corporation (Billerica, America). The intensity of the protein bands was determined by ImageJ software. GAPDH protein was used for normalization.