Amersham ecl western blot system

Prepared target cell protein was used to investigate the expression levels of apoptosis-related proteins using the human apoptosis antibody array (cat. no. ab134001; Abcam). After the membrane was blocked with array buffer at room temperature for 1 h, prepared HuCCT1 cell protein was incubated with the membrane at 4°C overnight. The array was subsequently washed, and incubated with detection antibodies (1:100; cat. no. ab134001; Abcam) and streptavidin-HRP (cat. no. ab134001; Abcam) at room temperature for 1 h. Protein expression was visualized using the Amersham ECL + TM Western Blot system (Cytiva) and signal intensities were analyzed using ImageJ software version 1.8.0.112.

Total protein obtained using RIPA lysis buffer (Beyotime, Jiangsu, China) from HCCC-9810 and RBE cells and quantified using a BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc.) and 20 µg/lane protein was separated by SDS-PAGE on a 10% gel [30% acrylamide/bis-acrylamide; 1.5 mol/l Tris (pH 8.8); 10% SDS; 10% ammonium persulfate and tetramethylethylenediamine]. The separated proteins were transferred onto PVDF membranes and blocked using TBST solution containing 5% skimmed milk and 0.5 ml Tween-20 at 4°C for 1 h. Immunostaining was performed using target primary antibodies: Anti-MCM8 (1:1,000; cat. no. PA5-41325; Invitrogen), anti-Akt, (1:1,000; product no. 4685; CST), anti-p-Akt (1:1,000; cat. no. BS-5193R; Bioss), anti-MAPK9 (1:1,000; product code ab76125; Abcam), anti-PIK3CA (1:1,000; product code ab40776; Abcam), GAPDH (1:3,000; cat. no. AP0063; Bioworld) at 4°C for 4 h and corresponding secondary antibody goat anti-rabbit (1:3,000; cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature for 2 h. Protein bands were visualized using the Amersham ECL + TM Western Blot system (Cytiva) and signal intensities were analyzed using ImageJ software version 1.8.0.112 (National Institutes of Health).