CHIP-T246M expressed from the endogenous locus results is regulated post-translationally resulting in decreased steady-state protein levels, however, interactions between CHIP and known interactors are not affected.

(A) Immunoblot analysis of CHIP and β-tubulin protein in primary fibroblasts (MEFs) isolated from T246/T246 (T/T), T246/M246 (T/M), or M246/M246 (M/M) mouse embryos (B) Maximum intensity projections of CHIP immunofluorescence in MEFs treated with 0.05% DMSO or MG132. (C) qPCR analysis of Stub1 mRNA levels in MEFs represented by the dot plot and summarized by the mean and 95% confidence intervals. (D) Immunoblot analysis of CHIP and β-actin protein in fibroblasts isolated from control patients (WT) or siblings that are homozygous for CHIP-T246M (II-1 and II-2). (E) Immunoblot analysis of CHIP under reducing SDS-PAGE (left) or blue native PAGE (right) of varying amounts of protein extracts (μg) from cerebellum (Cbl) or testes (Ts) from mice with the indicated genotypes. Protein loading on the reducing blot was confirmed via stain-free technology. As a reference, migration of recombinant CHIP protein is provided (purple). The dashed boxes identify the regions used for longer exposure. (F) Immunoblot analysis of CHIP and β-tubulin in soluble cell lysates from either T/T or M/M, fibroblasts, or fibroblasts isolated from CHIP−/− embryos (KO) after exposure to cycloheximide (CHX) indicated in hours (h). Two exposures of CHIP immunoblots are provided to help visualize M/M conditions. (G) Immunoblot analysis of CHIP and β-actin in either the soluble or insoluble fraction of lysates or whole cell lysates from T/T or M/M MEFs treated with 20 μM MG132 or 0.05% DMSO control for 4 hours. (H) Immunoblot analysis of AMPKα1 and CHIP in cell lysates from T/T or M/M MEFs either before (input) or after immunoprecipitation (IP) of the indicated protein. Control samples (C) contained a mixture of 50% T/T and T/M and were immunoprecipitated with either rabbit IgG or goat IgG as controls for the CHIP and AMPKα1 antibodies, respectively. (I) Immunoblot analysis of HSC70 and CHIP in cell lysates from T/T or M/M MEFs either before (input) or after immunoprecipitation (IP) of CHIP. Control samples (C) contained a mixture of 50% T/T and T/M and were immunoprecipitated with rabbit IgG to control for the CHIP antibody. (J) Immunoblot analysis of HSP70 and CHIP in MEFs with the indicated genotypes that were treated without heat shock (no) or with heat shock (HS) followed by the indicated recovery time. Densitometry of relative HSP70 protein is represented by the bar graph and 95% CI, N = 3; **, † correspond to p < 0.01 or 0.05 via Tukey’s post hoc test comparing M/M to either T/T or CHIP−/−, respectively, at the indicated time point.