FIGURE 9.
Interaction of Sec61 complex with BiP and anti-loop 7 antibodies, respectively. A, co-immunoprecipitation of BiP and Sec61 complex from BiP-His6-containing HeLa cell extracts was carried out as described under “Experimental Procedures.” The precipitates were analyzed for BiP and Sec61α, respectively. 40% of the HeLa cell extract that was used for the precipitation was analyzed in parallel (lane 1). Only the areas of interest from single gels are shown. B, pull-down of Sec61 complex from canine pancreatic microsomal extracts was carried out with anti-loop 7 antibodies as described under “Experimental Procedures.” Affinity-purified anti-Sec61β antibodies served as positive control (lane 4). The precipitates were collected with Dynabeads. Dynabeads without antibody served as negative control (lane 2). Molecular mass marker (M) (lane 1) and rough canine pancreatic microsomes (rough microsomes; RM) (lane 5) were subjected to gel electrophoresis in parallel. All samples were analyzed for Sec61α. Only the areas of interest from single gels are shown. C and D, current voltage relationship from bilayers containing two active Sec61 channels in the absence or presence of affinity-purified anti-Sec61β at 2.5 μg/ml final concentration on both sides of the bilayer. PKRM, rough microsomes, which were treated with puromycin plus high salt, were used as the source of the Sec61 channels. E and F, conductance histograms from single channel recordings obtained from bilayers with single active Sec61 channels, average of n = 3 recordings at Vm = +40 mV in the absence of and after 30-min incubation with loop 7 Fabs (final concentration, 2 μg/ml).