Western Blot Analysis of ZIP7 Phosphorylation

Cells were lysed by sonication using a Vibra Cell sonicator (Sonics & Materials, USA) and heated for 5 min at 95 °C. An equivalent of 3x10⁶ cells per lane was separated at 170 V on 10% polyacrylamide gels. After separation, samples were blotted onto nitrocellulose membranes (GE Healthcare Life Sciences, Germany). Uniform loading of gels was confirmed by Ponceau S (AppliChem, Germany) staining. Subsequently, membranes were blocked for 1 h with TBS-T (20 mM Tris–HCl [pH 7.6], 137 mM NaCl, 0.1% [v/v] Tween 20), containing 5% fat-free dry milk, and were washed afterwards thrice with TBS-T. Incubation with primary mouse monoclonal pZIP7 (S275/S276) [21] (link), ZIP7 (ProteinTech, United Kingdom) and β-actin (Cell Signaling Technology, Germany) antibodies was performed overnight at 4 °C at 1/1000 dilution in TBS-T, containing 5% BSA. Afterwards, membranes were washed thrice with TBS-T and incubated with either anti-rabbit-HRP (for β-actin and ZIP7) or anti-mouse-HRP (for pZIP7) (both from Cell Signaling Technology, Germany) secondary antibodies 1/2000 in TBS-T with 5% fat-free dry milk. After again washing thrice with TBS-T, immunodetection was performed using LumiGlo Reagent (Cell Signaling Technology, Germany) on LAS-3000 (Fujifilm Lifescience, Germany). Band density was determined with ImageJ software (NIH, USA).