Fig. 4

CIC interacts with the E3-ligase TRIM25. a Volcano plot showing CIC-interacting proteins identified using CIC immunoprecipitation followed by mass spectrometry in ATXN1L^KO NHA cells. Red data points are high confidence interactors. b Representative Western blot of CIC immunoprecipitation showing interaction with TRIM25 in ATXN1L^WT (NHA) and ATXN1L^KO (B82) cell lines. Right: barplot showing quantifications of TRIM25 Western blots. Quantifications were normalized to CIC Western blots. c Immunofluorescence images of proximity ligation assay showing FLAG-tagged CIC-S-TRIM25 interaction in ATXN1L^WT cells treated with ATXN1L siRNA. Scrambled siRNA was used as a negative control. White bars denote 10 μm. Right: Tukey boxplots showing quantification of the number of FLAG-TRIM25 foci/cell. d Representative Western blot of ATXN1L^WT (HEK) and ATXN1L^KO (A10, A30, B21) cell lines treated with TRIM25 siRNA. Scrambled siRNA was used as a negative control. Below: barplot quantifications of CIC protein expression. Quantifications were normalized to vinculin. e Relative mRNA expression of CIC and CIC target genes ETV1/4/5 following treatment with TRIM25 siRNA for 48 h. Scrambled siRNA was used as a negative control. Gene expression was normalized to TBP. f Representative Western blot of ATXN1L^WT (NHA) and ATXN1L^KO (B82) cell lines ectopically overexpressing FLAG-tagged TRIM25. Empty FLAG vector was used as a negative control. Western blot and RT-qPCR quantifications were collected from 3 independent experiments. PLA quantifications were collected from 65 individual cells. Error bars represent one standard deviation. p values were calculated using the two-tailed independent Student’s t test. Statistically significant values are denoted (*p < 0.05, **p < 0.01, ***p < 0.001). Individual data values can be found in Additional file 17: Table S10