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. 2012 Jun 14;287(32):26806–26816. doi: 10.1074/jbc.M112.376202

FIGURE 3.

FIGURE 3.

Cross-linking of ICL1 and ICL3 with M4M highly stimulates ATPase activity. A, model showing predicted orientations of Leu175, Asp177, and Asn820 in the closed conformation. B, membranes prepared from HEK 293 cells expressing the ICL1/ICL3 mutants D177C/N820C or L175C/N820C were treated with cross-linkers M1M, M4M, or M17M or no cross-linker (Control). The reactions were stopped by addition of SDS sample buffer containing no thiol-reducing agent and samples subjected to immunoblot analysis. The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. C, membranes expressing mutants D177C/N820C or L175C/N820C were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. The isolated P-gps were mixed with lipid and ATPase activities were measured in the absence (−) or presence (+) of 0.3 mm verapamil. In some cases the samples were treated with 10 mm dithiothreitol (+DTT) before assay. Each value is the mean ± S.D. (n = 3).