Fig. 1. Native and recombinant his-tagged lasp-1 co-sediment with purified non-muscle F-actin but GST-tagged lasp-1 does not co-sediment with G-actin in cell lysates. (A) Coomassie-blue-stained SDS-PAGE gel showing typical actin co-sedimentation assay. Lasp-1 (8 µM, arrow) or -actinin (2 µM, star, positive control) were incubated with actin (23 µM, arrowhead). After actin polymerization, supernatants (lanes 1-5) and pellets (lanes 6-10) were prepared and resolved by electrophoresis. Lanes 1,2,6,7: lasp-1 without F-actin. Lanes 3,4,8,9: lasp-1+F-actin. Lanes 5,10: -actinin+F-actin. Std: BioRad precision M_r standard (100, 75, 50, 37, 25 kDa). Note that his-tagged lasp-1 migrates with an apparent molecular mass of 38 kDa. Previous analyses with less precise M_r standards reported an apparent molecular mass of 41 kDa for his-tagged lasp-1 and 40 kDa for native lasp-1 (Chew and Brown, 1987; Chew et al., 1998). (B) Western blot of actin co-sedimentation of duplicate samples of endogenous lasp-1 in parietal cell extracts. Lasp-1 was co-sedimented with 7.5 µM F-actin as described in Materials and Methods. Similar results were obtained in three independent experiments. (C) Quantitation of lasp-1 association with F-actin at different concentrations of lasp-1. The F-actin concentration was 14 µM. Corrections for nonspecific precipitation of his-tagged lasp-1 were performed for each concentration. Values are means±s.e.m. for n=4 independent experiments. (D) Western blot of GST pull-down assay fractions using an actin antibody showing that similar amounts of actin in samples with GST-sepharose vs GST-tagged lasp-1. As expected, no signal was detected in the absence of parietal cell lysate. Similar results were obtained in four independent experiments. See Materials and Methods for details.