Fig. 6

CCDC74A/B possess self-association activity. a–c GST pull-down assays. Flag-CCDC74B (a, c) or CCDC74A-GFP (b) (expressed in HEK293T cells) and GST-CCDC74B full-length or mutants (expressed in E. coli and purified) were incubated and then analyzed by Western blotting with anti-Flag (a, c) or anti-GFP (b) antibody and by Coomassie blue (CBB) staining. d Flag-CCDC74B-expressed HeLa cells were treated with 1 mM disuccinimidyl suberate (DSS) in DMSO for 30 min, then the samples underwent Western blotting with anti-Flag antibody. The long time exposure showed a dense band of CCDC74B dimers. e Schematic of human CCDC74B domains, illustrating seven amino acids required for CCDC74B dimerization. f Flag-CCDC74B wild-type (WT)- or 7N mutant-expressed HeLa cells were treated with 1 mM DSS in DMSO for 30 min. Samples were analyzed by Western blotting with anti-Flag antibody. 7N: MLAL67-71NNNN and LLL230-234 NNN. g GST-CCDC74B WT- or 7N mutant-coupled glutathione sepharose 4B beads were incubated in vitro with assembled, taxol-stabilized microtubules in BRB80 buffer at room temperature. The bead-bound proteins were analyzed by Western blotting with anti-tubulin antibody and by CBB staining. GST proteins served as a negative control. h Relative intensity of tubulin bands in g. The signal from GST group was normalized to 1.0. Data are mean ± SEM (one-way ANOVA test, ***P < 0.001; n.s., not significant; three independent experiments)