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Fig. 2. HRSL3 (HREV1) interacts with the endogenous regulatory, but not the catalytic, subunits of PP2A. (A) Protein extracts from several cell lines were analyzed by immunoblotting against an HRSL3-specific antibody described previously as H-REV107-1. As a control, COS-7 cells transiently transfected with an HRASL3 expression plasmid, were used. PR36
and PR65 were detected with appropriate antibodies. Actin was used as a loading control. (B) The PR65 immunocomplex was recovered from OVCAR-3 cells transiently transfected with an HRASL3 expression plasmid (HREV1FL). Immunoprecipitated proteins (IP), and protein extracts used for the immunoprecipitation (Input) were subjected to SDS-PAGE and western blot analysis. Presence of the regulatory PR65 (top) and the catalytic PR36 (middle) subunits was demonstrated by immunoblotting with appropriate antibodies. Application of an anti-HRSL3 specific antibody (bottom) revealed the presence of the HRSL3 protein in the PR65 protein complex (second lane) but not in the control immunoprecipitation (third lane), confirming the interaction between HRSL3 and endogenous PR65. (C) OVCAR-3 cells were transiently transfected with HREV1FL, dNdC and pcDNA3 as a control. Forty-eight hours post-transfection immunoprecipitation was performed with a PR65-specific antibody (left panel) and with a PR36,
-specific antibody (right panel). Presence of the HRSL3 full-length and truncated proteins, PR65,, PR36, and B56 in the protein extracts used for immunoprecipitation (Input) and in the precipitated protein complexes was analyzed using western blotting with the appropriate antibodies. *Decreased amount of the PR36 proteins in the PR65 protein complex in the presence of HRSL3 was semiquantitatively evaluated (see bar chart below) using ImageJ freeware. The ratio between the amounts of immunoprecipitated PR36 and the light chain of the PR65 antibody is shown on top of the bars. (D) OVCAR-3 cells were transiently transfected with HREV1FL-V5 and incubated with PR65, and V5-specific antibodies. Then the cells were stained with DAPI to visualize nuclei (blue), with Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies to visualize the distribution of the PR65 (red) and the HRSL3-V5 (green) proteins, respectively using confocal microscopy. (Merge) Yellow color indicates a colocalization of the HRSL3 and the PR65 proteins (overlay of red and green) in the cytoplasm. (E) OVCAR-3 cells were transiently transfected with HREV1FL and incubated with PR36, and HRSL3-specific antibodies. Then the cells were stained with DAPI to visualize nuclei (blue), with Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies to visualize distribution of the PR36 (green) and the HRSL3 (red) proteins, respectively using confocal microscopy. (Merge) Absence of yellow color indicates no colocalization of the HRSL3 and the PR36 protein in the cells.
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