Figure 4

Differential levels of CHIP protein in fibroblasts from patients. Fibroblasts from Patient 1 (P1[II-1]) and Patient 2 (P2) show lower steady-state levels of CHIP protein compared with normal fibroblasts (WT), as analyzed by SDS-PAGE and immunoblotting using CHIP-specific antibody. In addition, the band corresponding to CHIP-N65S mutant in P1 reveals slightly faster migration rate on SDS-PAGE compared to the CHIP-WT band, probably due to protein conformational changes induced by the mutation. In fibroblasts from the father of Patient 1 (P1[I-1]), a heterozygous carrier of the N65S mutant allele, a double band can be observed (CHIP-WT and CHIP-N65S). In P2 a weak band most likely corresponding to CHIP-E28K is detected, while the band corresponding to the lower molecular weight CHIP-K144* form of ~16 kDa is not observed on this blot. Actin-specific protein bands are shown to compare the relative amounts of total protein loaded per lane.