Pdgfra

ACSL4, SAR1A, PDE7B, PDGFRA, and MAP2K1 were detected by western blot analysis following the overexpression of miR-34c or siRNA. Cells were lysed using M-PER Protein Extraction Reagent (Pierce, USA) supplemented with a protease inhibitor (PMSF). Protein concentrations of the extracts were measured with the BCA assay (Pierce, USA) and equalized with extraction reagent. Equal amounts of extracts were loaded and subjected to SDS-PAGE, followed by transfer onto nitrocellulose membranes. Primary antibodies (ACSL4, 1:500; SAR1A, 1:500; PDE7B, 1:800; PDGFRA, 1:500; and MAP2K1, 1:200) and horseradish peroxidase-coupled secondary antibodies (1:2000) were purchased from Abcam, USA. Membranes were probed using ultra-enhanced chemiluminescence western blotting detection reagents. GAPDH was used as an internal control. Protein abundance was analyzed using ImageJ tools.

Cell lysis was done in chilled RIPA lysate buffer (Beyotime, China) with protease inhibitor and phosphatase inhibitor. Equal amounts of total protein were separated on SDS-PAGE gels at 100 V for 1.5 h, before transfer to 0.45 μm PVDF membrane (NEST, China). Blocking was done in 5% non-fat dry milk in TBST with subsequent overnight incubation with primary antibodies at 4 °C. Primary antibodies were against PDGFRA (1:1000, abcam), p-PDGFRα (1:1000, abcam),β-tubulin (1:1000, wanleibio, China), GAPDH (1:5000, abcam), vimentin (1:500, wanleibio, China), E-cadherin (1:500, wanleibio, China), ZEB1 (1:500, wanleibio, China), GPX4 (1:1000, abcam), NRF2 (1:1000, abcam) and SLC7A11 (1:1000, abcam). Upon three TBST washes, membranes were incubated with secondary antibody, before visualization with enhanced chemiluminescence (ECL) reagent (Yeasen, Shanghai, China).