Paxillin

Western-blot assays were used to analyze protein expression [44 (link)]. The following antibodies were used: DDX17 (Proteintech, Cat. 19910-1-AP), MAGEA6 (SAB, Cat. 43096), p62 (Proteintech, Cat. 18420-1-AP), LC3 (Sigma, Cat. L8918), β-actin (Proteintech, Cat. 81115-1-RR), GAPDH (Proteintech, Cat. 10494-1-AP), AMPKα1 (Abcam, Cat. ab32047), paxillin (Proteintech, Cat. 10029-1-Ig), GFP (Proteintech, Cat. 66002-1-Ig), MYL9 (Abcam, Cat. ab191393) and the HRP-conjugated goat anti-rabbit/mouse secondary antibody (Jackson ImmunoResearch, PA, USA).

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).