The prefoldin complex stabilizes the von Hippel-Lindau protein against aggregation and degradation
Fig 1
Prefoldin subunits PFDN1, PFDN2 and PFDN3 associate with pVHL in human cells.
A) Scheme depicting the different BirA fusion proteins used in the BioID experiment (ex1, ex2 and ex3 refer to VHL exon1, exon2 and exon3-encoded polypeptides, respectively), B) Western blot analysis of PFDN1, PFDN2, PFDN3 and BirA fusion proteins in total protein extracts before (Input) and in fractions recovered after the Streptavidin affinity-chromatography column (Bound). Cullin 2 (CUL2) and Elongin C (ELOC) were used as positive controls whereas GAPDH and p44/42 ERK were used as negative controls. Ctl corresponds to untransfected control cells. Left: HEK293 cells and right HeLa cells. C) Quantification of the biotinylated prefoldin / pVHL expression levels to map the prefoldin binding site in pVHL ORF: truncated VHL213-BirA constructs expressing either all 3 exons (VHL213), exons 1+3 (VHL172), exons 1+2 (VHLex1&2) and exons 2+3 (VHLex2&3) were used in a BioID experiment in HeLa and 786-O cells. Histograms represent the mean relative levels of recovered PFDN1 (upper panel) and PFDN3 (lower panel) proteins. Full-length VHL213-BirA level was set as 1. D) HeLa cells expressing GFP, VHL213-GFP (213), VHL172-GFP (172) or control untransfected cells (Ctl) were lysed and immunoprecipitation was performed with GFP-trap. The whole cell lysates (Input) and immunoprecipitates (Bound) were analyzed by Western blot using anti-GFP, anti-CUL2, anti-PFDN1, anti-PFDN3 and anti-PFDN5 antibodies.