Discovering a protein's 'elevator motion' could spur new cancer treatments NatureComms
cells expressing BbZIP variants were incubated with 3 mM NEM for 1 h at 4 °C, and then washed twice to remove excessive NEM with the buffer containing100 mM Tris, pH 7.0, 60 mM NaCl, 10 mM KCl through centrifugation. The membrane fraction was then collected and dissolved with the denaturing buffer containing 6 M urea, 0.5% SDS, and 0.
To test the effects of metal substrate on cysteine accessibility, the selected variants in the native membrane were subjected to NEM titration with indicated concentrations in the presence and absence of 50 µM ZnClCysteine accessibility assay was also performed using mPEG5K as the thiol reacting reagent. Selected purified proteins at 10–20 µM were treated with 1 mM mPEG5K for 0 or 30 min in the presence of 3 mM EDTA.
. An HRP-conjugated anti-mouse immunoglobulin-G antibody was used as the secondary antibody at 1:5000 dilution in Western blot.Human embryonic kidney cells were cultured in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum and Antibiotic-Antimycotic solution at 5% COand 37 °C. Cells were seeded on the polystyrene 24-well trays for 16 h in the basal medium and transfected with 0.8 μg DNA/well using lipofectamine 2000 in DMEM with 10% FBS.
The zinc transport activities of ZIP4 and the variants were tested using the cell-based transport assay. Twenty hours post-transfection, cells were washed with the washing buffer followed by incubation with Chelex-treated DMEM media . 5 μM Zn was added to cells. After incubation at 37 °C for 30 min, the plates were transferred on ice and the ice-cold washing buffer with 1 mM EDTA was added to stop metal uptake.
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