Effects of zinc and hypoxia on nuclear localization and DNA binding of MTF-1. (A) C 2 C 12 cells were untreated (control) with 100 μmol/L ZnCl 2 , or exposed to hypoxia (pO 2 = 1%) for 16 h, as indicated. Extracts were analyzed by electrophoretic mobility shift assays (EMSA) using labeled MRE-s oligos. Only the region of the gel containing the specific MTF-1 complexes is shown. (B) Immunoblotting detection of MTF-1 in whole-cell (WCE), nuclear, and cytosolic extracts. Extracts were prepared from L cells that were untreated (0), treated with 100 μmol/L ZnCl 2 (Zn), or exposed to hypoxia (Hx) (pO 2 = 1%), as indicated, and analyzed by immunoblotting with antibodies against MTF-1 and tubulin. Because tubulin is not an appropriate loading control for nuclear extracts, a particular band detected on the blot by staining with amido black is shown. The results shown in panels A and B are representative of 3 independent experiments. (C) ChIP assays were performed using chromatin isolated from L cells that were untreated or treatedwith 100 μmol/L ZnCl 2 for 3 h or exposed to hypoxia for 16 h, prior to formaldehyde crosslinking. Immunoprecipitation of crosslinked chromatin was done with MTF-1 (anti-MTF-1) or HIF-1α (anti-HIF1α) antibodies or a pre-immune normal rabbit serum (PI), as indicated. DNA from both the IP input (prior to immunoprecipitation) and the IP-bound fractions was amplified by PCR with primer pairs for the mouse Mt-1 or Glut-1 promoter. Input, amplification of DNA prior to immunoprecipitation. The input sample contained 0.4% of the supernatant used for immunoprecipitation of crosslinked MTF-1. The PCR products were analyzed by agarose gel electrophoresis. These ChIP assays were performed 3 times using 3 different chromatin preparations with similar results.
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