Fig.S15

Fig.S15. (A) Representative Immunoblot for the analysis of endogenous SPOUT1/CENP-32 levels upon Control (siRNA C) or SPOUT1/CENP-32 depletion (siRNA C32) using siRNA oligonucleotides (left panel) and the corresponding tubulin signal as a loading control (right panel). (C-E) Representative Immunoblots of SPOUT1/CENP-32-GFP constructs (CENP-32-GFP WT, CENP-32-GFP N86D, CENP-32-GFP G98S, CENP-32-GFP G244S, CENP-32-GFP T289M, CENP-32-GFP G293S, CENP-32-GFP T353M or CENP-32-GFP A356N) and the corresponding tubulin signal as a loading control, showing comparable expression levels among all inducible cell lines. Quantification of SPOUT1/CENP-32/Tubulin ratio using uncalibrated OD values (E) was performed with ImageJ. Three independent experiments; mean ± SEM; non-parametric ANOVA with Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (F) Representative fluorescence images of a Control (siRNA C) or SPOUT1/CENP-32 siRNA (siRNA C32) rescue assay in the stable U2OS cell lines with inducible expression of SPOUT1/CENP-32 WT-GFP and SPOUT1/CENP-32 A356N-GFP. Conditions without doxycycline from Fig. 6C. Scale bar, 10 um.