Anti pik3ca

After transfection or treatment, the cells were lysed using RIPA lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) supplemented with a Protease Inhibitor Cocktail (Pierce; Thermo Fisher Scientific, Inc.). Then, the protein concentration of the lysates was determined using the BCA method (Pierce; Thermo Fisher Scientific, Inc.). Following this, the protein solution was boiled with equivalent amounts of loading buffer for 10 min, equal amounts of protein (30 µg) were subjected to 8–12% SDS-PAGE gel electrophoresis, and then transferred onto a PVDF membrane (Millipore). Membranes were blocked with 5% non-fat milk in Tris-buffered saline and incubated with anti-PIK3CA (product code ab40776; dilution 1:1,000), PI3K (product code ab32089; dilution 1:1,000), Akt (product code ab179463; dilution 1:1,000), phosphorylated (p)Akt (product code ab81283; dilution 1:5,000), or GAPDH (product code ab181602; dilution 1:5,000; Abcam) antibodies at 4°C overnight followed by horseradish peroxidase-conjugated secondary antibody (product code ab205718; dilution 1:10,000; Abcam). Subsequently, the protein bands in the membranes were visualized using the ECL method (Pierce; Thermo Fisher Scientific, Inc.) The gel optical processing system (Image-Pro Plus 6.0; Media Cybernetics) was used to analyze the net optical density of the bands.

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).

The cells were lysed with RIPA buffer supplemented with protease inhibitors. Total protein extract was separated on 12% SDS-PAGE gels and then transferred to nitrocellulose (NC) membranes. The membrane, after blocked by 5% fat-free milk, was incubated with primary antibodies at 4 °C for 12 hrs. The primary antibodies included anti-PIK3CA (1:1000, Abcam, ab40776), anti-E-cadherin (1:1000, Abcam, ab1416), anti-N-cadherin (1:1000, Abcam, ab18203), anti-Vimentin (1:1000, Abcam, ab92547), anti-ZO-1 (1:1000, Abcam, ab96587) and GAPDH (1:1000, Abcam, ab8245). Following that, the NC membrane was then rinsed with TBST solution and incubated at room temperature for 1 hr with the secondary antibodies (1:2000, Santa Cruz Biotechnology) conjugated to HRP. After that, color rendering was performed using hypersensitive ECL (Hubei Biossci Biotechnology Co, Ltd.). The automatic developing instrument (ChemiDocXRS imaging system) was adopted to detect the signal.