Clarity western ecl substrates

Total proteins from cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime). Protein concentrations were determined using the Pierce BCA protein assay kit (23227, Thermo). Aliquots of 20 μg proteins were separated on a 10% SDS‐PAGE gel and electro‐transferred onto a polyvinylidene difluoride (PVDF) membrane (ISEQ00010, Merck Millipore). After blocking using 5% skim milk, the membrane was incubated with primary antibody at 4°C overnight and subsequently with the corresponding secondary antibody (Santa Cruz Biotech) for 1 h at room temperature. The membrane was then developed using Clarity Western ECL substrates (WBKLS0500, Merck Millipore) and visualized with a ChemiDoc MP Imaging System (Bio‐Rad). Protein expression was normalized with β‐tubulin or β‐actin expression. The antibodies against LPAR1 (#3U1Q0), β‐tubulin (2G10) and FAP (F11‐24) were purchased from Invitrogen. The antibodies against α‐SMA (D4K9N) and β‐actin (8H10D10) were purchased from Cell Signaling Technology. The antibodies against B7‐H3 (#376769) were purchased from Santa Cruz Biotech.