Fig. 1. (A) Western blot analysis with recombinant protein fragments expressed in E. coli BL21DE3 cells. BL21DE3 cells not expressing exogenous protein (BL21) or expressing the p0071 tail (p0071), the NPRAP/-catenin tail (NPRAP), the p120^ctn tail (p120^ctn) or the plakophilin 1 repeat + tail domains (PKP1) were lysed in SDS sample buffer, separated on 15% gels and transferred to membranes. Membranes were probed with 6D1-10-, p120^ctn- and His-tag monoclonal antibodies as indicated. 6D1-10 antibody recognized NPRAP and p0071, but not p120^ctn or plakophilin 1, the p120^ctn antibody was specific for the p120^ctn tail and the His-tag antibody detected all recombinant proteins. (B) Total cell extracts from kidney were prepared in SDS sample buffer, separated on 7% gels and transferred to membranes. Individual lanes were probed with antibodies to p0071 (6D1-10), p120^ctn and plakophilin 1, 2 and 3, as indicated. 6D1-10 detected a single band of 135 kDa that differs from protein bands detected by p120^ctn- and plakophilin antibodies. (C) Intracellular localization of endogenous p0071 in MCF-7 cells as detected by antibody 6D1-10. MCF-7 cells were extracted with Triton-X 100 prior to fixation and double labeled with the 6D1-10 monoclonal antibody (p0071) and rabbit anti-desmoplakin (serum 616) and rabbit polyclonal anti-Pan-cadherin (cadherin). Plasma membrane regions denoted by arrows are shown in detail at higher magnification. Bars, 3 µm.