Fig. 5

The CHIP mutations abolish the interaction between E2 ubiquitin ligase and CHIP, enhance caspase-3 cleavage and alter cerebellum development. A SH-SY5Y cells were transfected with the CHIP WT, p.Glu278fs, or Δ278–303 plasmid for 48 h. Immunoprecipitations were performed using an anti-FLAG antibody. Immunoprecipitates were sequentially probed with anti-UBE2D1, anti-UBE2D2, anti-UBE2D3, and anti-FLAG antibodies. Five percent of lysates used for immunoprecipitation were loaded as the inputs and probed with anti-UBE2D1, anti-UBE2D2, anti-UBE2D3, and anti-FLAG antibodies. IgG served as an IP negative control, and β-actin as a loading control. B SH-SY5Y cells were transfected with the CHIP WT, p.Glu278fs, or Δ278–303 plasmid for 48 h and subjected to Western blot analysis. Cleaved caspase-3 was detected. β-actin served as a loading control. C SH-SY5Y cells were transfected with the plko.1-puro empty vector, plko.1-puro scramble shRNA and plko.1-puro STUB1 shRNA plasmid for 48 h and subjected to Western blot analysis. CHIP was detected to examine the knockdown efficiency of plko.1-puro STUB1 shRNA. β-actin served as a loading control. (D) Mouse cerebellum was electroporated with CHIP cDNA or shCHIP along with GFP at P6 and dissected 2 days later. The upper panel shows the distribution of electroporated GFP + GNPs (green). The lower panel shows the staining of the cell cycle marker Ki67 (red). Brain slices were stained with the DNA dye, DAPI (blue). Arrows: GFP + , Ki67 + cells. E Bar graph showing the distribution of GFP + cells in different layers. In the control cerebellum, about half the GNPs migrated from the EGL to the ML and IGL. Overexpression of CHIP arrested cells mostly in the oEGL, while knockdown of CHIP arrested cells mostly in the iEGL. F Bar graph showing the percentage of Ki67 + cells among all GFP + cells. CHIP overexpression leads to a dramatic increase in the percentage of Ki67 + cells. (***p < 0.001, **p < 0.002, n = 3 animals, one-way ANOVA.)