Fig. 5.
Saturation of the aggregon induces puncta formation of conductin. a Schematic illustration of conductin RGS aggregation with inhibited DIX-mediated polymerization, which can be shifted towards high-order DIX-mediated polymerization by (i) QV-PS mutation (red x), (ii) RGS co-expression, or (iii) the small peptide P182–195 (red triangle). b Immunofluorescence staining for Flag (red) in U2OS cells transfected with Flag-tagged conductin (Flag-Cdt) either together with conductin 2–345 or conductin 2–345 QV-PS tagged with GFP (green). Scale bar: 20 µm. c Percentage of transfected cells exhibiting Cdt puncta. Per bar, 1500 cells of three independent experiments as in b were analyzed. Results are mean ± SEM (n = 3). d Western blotting for indicated proteins in U2OS cell extracts. e Immunofluorescence staining of conductin or its M3 mutant in U2OS cells transfected with HA-tagged conductin or conductin M3 either alone, or together with P182–195, or the QV-PS mutant of the peptide. Scale bar: 20 µm. f Percentage of transfected cells exhibiting Cdt or CdtM3 puncta. Per bar, 900 cells of three independent experiments as in e were analyzed. Results are mean ± SEM (n = 3). g, j Dot blot: Detection of GFP(-tagged) proteins binding to membrane pieces which were spotted with H2O (−) or 8 nmol of R9, P182–195-R9 or the QV-PS mutated peptide (QV-PS-R9) prior to incubation with lysates of HEK293T cells transfected with indicated plasmids (g) or with in vitro translated GFP-RGS (j). h Western blotting for GFP in the lysates used in g. Loading control: α-tubulin. i, l 2D densitometry quantification of dot blots in g and j, respectively. Results are presented relative to the GFP/R9 (i) or the GFP-RGS/R9 (l) combination as mean ± SEM of five independent dot blots (n = 5). k Coomassie Brilliant Blue staining of in vitro translated GFP-RGS (+DNA template lane, arrowhead) used in j. Bands also present without template DNA (−) show purified kit components. m Western blotting for HA under native conditions in lysates of HEK293T cells transfected with HA-Cdt 2–345 alone (−) or together with P182–195. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test). Source data are provided as a Source Data file