Bladder cancer cells were washed with ice‐cold phosphate buffered saline (PBS), lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and the concentration was measured by BCA Protein Assay Kit (Invitrogen).
18 (link),
19 (link) For western blot analysis, 30 μg of proteins were loaded onto 10% SDS‐PAGE and transferred to PVDF membranes. Next, the membranes were blocked with 5% milk for 1 h at room temperature, and incubated with anti‐FLRT2 (#ab154023, Abcam), anti‐ACSL4 (#ab155282, Abcam) or anti‐β‐actin (#ab8226, Abcam) overnight at 4°C. On the next day, the sample was then washed thrice with TBST for 10 min each time. The membrane was removed and incubated with the peroxidaseconjugated secondary antibody (ProteinTech Group) at 37°C for 2 h. Finally, ECL (Biosharp Life Sciences) colour was developed with β‐actin as an internal reference to analyse the protein expression level on the membrane and visualized with the by the chemiluminescence system (ChemiDocTM Touch; Bio‐Rad).
18 (link),
19 (link) For western blot analysis, 30 μg of proteins were loaded onto 10% SDS‐PAGE and transferred to PVDF membranes. Next, the membranes were blocked with 5% milk for 1 h at room temperature, and incubated with anti‐FLRT2 (#ab154023, Abcam), anti‐ACSL4 (#ab155282, Abcam) or anti‐β‐actin (#ab8226, Abcam) overnight at 4°C. On the next day, the sample was then washed thrice with TBST for 10 min each time. The membrane was removed and incubated with the peroxidaseconjugated secondary antibody (ProteinTech Group) at 37°C for 2 h. Finally, ECL (Biosharp Life Sciences) colour was developed with β‐actin as an internal reference to analyse the protein expression level on the membrane and visualized with the by the chemiluminescence system (ChemiDocTM Touch; Bio‐Rad).
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