PP1α isoform-specific antibody immunoprecipitation and detection of endogenous and induced PP1α by western blotting. PP1α isoform-specific antibody was used to immunoprecipitate proteins from equal quantities (50 ug) of uninduced (first lane from the left, top panel) and induced LLWO2F cell lysate (second lane from the left, top panel). The precipitated proteins were then separated by SDS-PAGE and immunoblotted with the same isoform-specific antibody. The positions of 6His-HA-PP1α and PP1α are indicated to the left of the top panel. Whole-cell lysates (10 ug) were separated in the next two lanes. Last lane contains 1 ug of purified recombinant PP1α as a control for endogenous PP1α (37 kDa). Bottom panel shows identical experiment using antibody to hemagglutinin for immunoprecipitation and immunodetection. Although low levels of 6His-HA-PP1α can be detected in cell lysate from uninduced cells following western blotting, this protein is almost undetectable in the same lysate following immunoprecipitation and western blotting. This observation is most likely due to low efficiency of immunoprecipitation.