Figure 5
CHIP-N65S causes a migration shift when analyzed by SDS-PAGE. (A) CHIP-WT and CHIP-N65S were translated in a TNT coupled transcription/translation system in the presence of [35S]Met, as described in Material & Methods. Samples were analyzed by SDS-PAGE and autoradiography. A double band can be observed for CHIP-WT, in which the lower band migrates at the same rate as CHIP-N65S. CTL; empty vector as negative control. (B) CHIP expression of transfected HEK293 cells with CHIP-WT or CHIP-N65S, tagged with V5 and His, and detected by SDS-PAGE and immunoblotting using anti-CHIP. Endogenous CHIP is observed at 35 kDa. The shift is observed only for transfected CHIP. A secondary isoform of CHIP appears at 32 kDa, lacking the first 72 amino acids, only observed in vitro[9]. CTL; empty vector as negative control. (C) Recombinant WT and CHIP variants expressed and purified from E. coli as MBP-fusion proteins and cleaved by TEV protease as described in Materials & Methods. Samples were analyzed by SDS-PAGE and coomassie staining. The migration shift is only observed for CHIP-N65S. CHIP-K144* appears at a lower molecular weight (truncated version of 16 kDa).