Ab126250

Proteins from cells were isolated using RIPA buffer (Vazyme) and the concentration was checked by Detergent Compatible Bradford Protein Quantification Kit (Vazyme). Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to segregate proteins and then the proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes (Vazyme). After being blocked with 5% skimmed milk (Vazyme) and washed by phosphate-buffered saline (PBS), the membranes were incubated with the primary antibodies: E-cadherin (1:3000, ab40772, Abcam, Cambridge, United Kingdom), Vimentin (1:2500, ab92547, Abcam), YAP1 (1:3000, ab52771, Abcam), C3aR (1:1000, ab126250, Abcam), ICAM-1 (1:1000, ab2213, Abcam), or β-actin (1:2500, ab8227, Abcam) overnight. After being rewashed with PBS, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 3 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad, Richmond, CA, USA) after being treated with ECL kit (Vazyme).

Total protein was extracted via RIPA buffer, which was evaluated by BCA kit (Beyotime). Samples were separated by SDS-PAGE and transferred onto PVDF membrane. Following being blocked, membrane was incubated by anti-C3AR1 (1:1,000; ab126250; Abcam, USA) or anti-GAPDH (1:1,000; ab8245) at 4°C overnight, followed by being probed with secondary antibody (1:5,000; ab7090). The protein bands were then visualized through ECL Plus substrate (Beyotime).