P paxillin

MDSCs were lysed in RIPA buffer (Beyotime Biotechnology), according to the manufacturer’s instruction. The protein samples were resolved on an SDS-polyacrylamide gel and then transferred to a PVDF membrane (Millipore Corporation). The membrane was incubated with a primary antibody against PEAR1 (Abbkine), MYOG (Santa Cruz), GAPDH (Proteintech), MYH3 (Santa Cruz), p-FAK (Bioss), FAK(Bioss), p-paxillin (Bioss), paxillin (Bioss), vinculin (Bioss), or ITGB1 (Bioss), followed by addition of a secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody (Bioss). The proteins were visualized by the Super ECL Plus detection kit (Applygen Technologies Inc) according to the manufacturer’s instructions. Images were acquired using the mini chemiluminescent imaging and analysis system MiniChemi™ 500 (Sage Creation Science).

Cells were homogenized and lysed in RIPA buffer (Beyotime, China) supplemented with proteinase inhibitors (Solarbio, China) and phosphatase inhibitors (NCM Biotech, China). Protein quantification was performed with the BCA assay (Beyotime, China) and equal amounts of proteins (30 μg) were separated and run on 10%–15% SDS‐PAGE gel following electrophoresis at 80 V for 2.5 h, then transferred onto the polyvinylidene fluoride (PVDF; Millipore, USA) membranes at 250 mA for 2.5 h. After blocked with 5% milk in 1 × TBST for 1 h, membranes were incubated with antibodies overnight at 4°C with the primary antibodies: SphK1 (1:1000, Abcam), LC3 (1:1,000, MBL), paxillin (1:1000, Proteintech), MMP2 (1:1000, Abclonal), p‐paxillin (1:800, Bioss), E‐cadherin (1:1000, CST), vimentin (1:1000, CST), and secondary antibody at room temperature for 1 h in lucifugal environment. GAPDH (1:10000, SAB) was used as the loading control. Images and band intensity were quantitated with Odyssey CLx Infrared Imaging System (LI‐COR Biosciences, Lincoln, NE, USA).