Slc39a7

Western blotting was performed as previously described 17 (link). Briefly, the total proteins of glioma cells or tissues were isolated using a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China). Protein lysates were transferred onto PVDF membranes after electrophoresis and blocked with 2% bovine serum albumin (KeyGen Biotechnology). The primary antibodies against SLC39A7 (1:1000; Abcam), TNF-α (1:1000; Abcam), p-p65 (1:1000; Cell Signaling Technology, Danvers, MA, USA), p65 (1:1000; Cell Signaling Technology), p-IκBα (1:1000; Cell Signaling Technology), IκBα (1:1000; Cell Signaling Technology), p-IKKα/β (1:1000; Cell Signaling Technology), IKKα (1:500; Cell Signaling Technology), IKKβ (1:500; Cell Signaling Technology) and β-actin (1:2000; ProteinTech, Chicago, IL, USA) were incubated at 4 °C overnight. After secondary antibody (ProteinTech) incubation, the bands were detected using a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) and quantified by Image J software (National Institutes of Health, Bethesda, MD, USA).

The detailed method of Western blot assay was described in our previous publication [23 (link)]. Briefly, MCF7 and MDA-MB-468 cells were transfected with siRNA-SLC39A7 or the control siRNA for 48 h. Cells were rinsed, harvested and dissolved in RIPA lysis buffer (Beyotime Institute of Biotechnology, China) with 0.5% cocktail protease inhibitor (Roche Diagnostics). Cell lysates were incubated on ice for 10 min, collected and sonicated for 15 s. Protein concentration was measured by BCA assay (BioRad Laboratories, U.S.A.). Protein was balanced, mixed with 5× loading buffer [Chunfeng Lv, China] and boiled for 5 min. Lysates were run 10% SDS-PAGE gels then transferred to PVDF membrane. After blocking with 5% milk in PBS (0.01% Tween 20) for 1 h, membranes were hybridized to primary antibodies overnight at 4°C. The primary antibodies used were as follows: GAPDH (cat no. 5174; 1:2000; Cell Signaling Technology, Inc; U.S.A.); SLC39A7 (cat no. ab254566; 1:1000; Abcam, U.K.).

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.