Western Blot Analysis of Transfected A549 Cells

After 48 h transfected with siRNA, A549 cells were undergone for a Western blot assay. [40 (link), 41 (link)] Cells were collected and homogenized in modified RIPA lysis buffer (Beyotime, China) with 0.5% protease inhibitor cocktail (Roche Diagnostics) and stayed on ice for 10 min. After centrifuging at 12,000 rpm, the supernatants were obtained and sonicated for 15 sec. The supernatants were quantified by BCA assay (BioRad Laboratories, USA). Then, the extracts were boiled with 5X loading buffer [Chunfeng Lv, China]. Twenty 20 μg total protein were electrophoresed through 10% SDS-PAGE gels and, then transferred onto the PVDF membrane. After blocking nonspecific binding sites with 5% milk in PBS for 1 h, membranes were incubated with primary antibodies of GAPDH (cat no. 5174; 1:2,000; Cell Signaling Technology, Inc; USA); RRBP1 (cat no. ab224354, 1:1000, Abcam, UK) or SLC39A7 (cat no. ab254566; 1:1000; Abcam, UK) overnight at 4° C. Then, the membranes were cultured with secondary antibodies conjugated with horseradish peroxidase at room temperature for 1 h. Last, the membranes were treated with ECL reagent for developing films at darkness.