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. 2017 Dec 31;9(12):10185–10202. doi: 10.18632/oncotarget.23796

Figure 2. Correlation of total and 1-phosphohitidine NME level in cell lysates.

Figure 2

A. Cell lysates prepared from vector and NME overexpression (NME1, NME2 or mouse ortholog Nme1) in MDA-MB-231T cells were assessed for 1-phosphohistidine (1-pHis) level using anti-N1-phosphohistidine antibody on a basic gel with no heating. For specificity, replicate samples were heated at 95°C for 10 min and loaded next to each sample (red). B. MDA-MB-435 cells lysates from vector (C100) and NME overexpression (H1-177) were also assessed for 1-pHis in similar manner. Following stripping, both blots were re-probed with total NME and β-actin antibodies. C. For immunofluorescence, MDA-MB-231T cells with GFP co-expressing vector or NME overexpression were fixed in PFA and stained for 1-pHis using anti-N1-phosphohistidine antibody. Following 1-pHis primary incubation, cells were washed twice and were then stained with total NME1/2 antibody. Nuclei were visualized using DAPI (Blue). Images were captured at 65x magnification. Yellow arrows indicate vacuoles in 1-pHis staining highlighting acidic compartments.