Anti acsl1

Western blotting analysis was performed as described previously (27 (link)). Briefly, the cells were lysed with 1% SDS lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Apexbio, Houston, USA). The protein concentration was determined by using a BCA protein assay reagent kit (Thermo Scientific). The protein content (18 μg) was separated by SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked overnight by 5% fat free milk in TBST at 4°C, followed by incubation at 4°C for 12 h with primary antibodies for anti-β-actin (1:5000) (Sigma), anti-β-Tubulin (1:5000) (TransGen Biotech), anti-ACSL1 (1:1000) (Cell Signaling Technology), ACC (1:1000) (Cell Signaling Technology), HK2 (1:1000) (Cell Signaling Technology), ACAA1 (1:1000) (Cell Signaling Technology), E-cadherin (1:1000) (Bioworld), N-cadherin (1:1000) (Bioworld), Twist1 (1:1000) (Cell Signaling Technology), respectively. The membranes were washed with TBST and incubated for 1.5 h with the corresponding HRP-conjugated secondary antibodies. The bands were visualized with ECL reagents (Merck, Billerica, MA, USA). The western blots were analyzed with the Image Lab Software (BIO-RAD, USA), and the program included an application with protein gels; the image exposure time to was set to 16 s.