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. 2014 Jan 1;25(1):184–195. doi: 10.1091/mbc.E13-06-0306

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© 2014 Bowe et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — PDLIM2 interacts with CSN subunits CSN5 and CSN8 and regulates CSN5 localization. (A) CSN5 immunoprecipitates from DU145 shScramble cells were assessed by Western blotting for PDLIM2 interaction. Beads and lysate without antibody (–Ab) is a negative control. (B) Western blotting for CSN5 and CSN2 expression in cytoplasmic (CY) and nuclear (N) extracts of DU145 shScramble or shPDLIM2 cells. Loading controls are tubulin (CY) and PARP (N). (C) Subcellular localization of CSN5 was assessed in shScramble and shPDLIM2 DU145 cells by immunostaining. Scale bars, 10 μM. (D) Peptide arrays were generated for all CSN subunits and overlaid with lysate from DU145 shScramble and DU145 shPDLIM2 cells. Spots that showed less reactivity with the anti-PDLIM2 antibody on PDLIM2 lysates were considered positive. CSN5 and CSN8 arrays showed intense spots of interaction with PDLIM2. Positive peptides were then synthesized with successive amino acid substitution (bottom, with graph and substitution arrays). Graphs represent intensity of interaction of peptides with PDLIM2 expressed as percentage of the intensity of the original interaction peptide. Alanines were substituted by glutamic acids; all other amino acids were substituted with alanines. (E, F) Immunoprecipitations (IPs) for CSN5 (E; top), PDLIM2 (E; bottom), or CSN8 (F), were performed from lysates of MCF7 cells stably transfected with GFP-empty vector (EV) or GFP-PDLIM2 as described in Materials and Methods. Immunocomplexes were probed for each protein by Western blotting. Controls for IPs included no lysate (–Ly) and beads plus lysate only, no antibody (–Ab). See also Supplemental Figure S4 and Supplemental Table S5.