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Fig.S15 Representative Immunoblots for the analysis of endogenous SPOUT1/CENP-32 and SPOUT1/CENP-32 constructs

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Description

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.

Data Citation

Abad, Maria Alba (2024). Fig.S15. figshare. Journal contribution. https://doi.org/10.6084/m9.figshare.26841688.v1
Date made available27 Nov 2024
PublisherFigshare

  • RNA methyltransferase SPOUT1/CENP-32 links mitotic spindle organization with the neurodevelopmental disorder SpADMiSS

    Dharmadhikari, A. V., Abad, M. A., Khan, S., Maroofian, R., Sands, T. T., Ullah, F., Samejima, I., Shen, Y., Wear, M. A., Moore, K. E., Kondakova, E., Mitina, N., Schaub, T., Lee, G. K., Umandap, C. H., Berger, S. M., Iglesias, A. D., Popp, B., Abou jamra, R. & Gabriel, H. & 62 others, Rentas, S., Rippert, A. L., Gray, C., Izumi, K., Conlin, L. K., Koboldt, D. C., Mosher, T. M., Hickey, S. E., Albert, D. V. F., Norwood, H., Lewanda, A. F., Dai, H., Liu, P., Mitani, T., Marafi, D., Eker, H. K., Pehlivan, D., Posey, J. E., Lippa, N. C., Vena, N., Heinzen, E. L., Goldstein, D. B., Mignot, C., De sainte agathe, J.-M., Al-Sannaa, N. A., Zamani, M., Sadeghian, S., Azizimalamiri, R., Seifia, T., Zaki, M. S., Abdel-Salam, G. M. H., Abdel-Hamid, M. S., Alabdi, L., Alkuraya, F. S., Dawoud, H., Lofty, A., Bauer, P., Zifarelli, G., Afzal, E., Zafar, F., Efthymiou, S., Gossett, D., Towne, M. C., Yeneabat, R., Perez-Duenas, B., Cazurro-Gutierrez, A., Verdura, E., Cantarin-Extremera, V., Marques, A. D. V., Helwak, A., Tollervey, D., Wontakal, S. N., Aggarwal, V. S., Rosenfeld, J. A., Tarabykin, V., Ohta, S., Lupski, J. R., Houlden, H., Earnshaw, W. C., Davis, E. E., Jeyaprakash, A. A. & Liao, J., 17 Feb 2025, In: Nature Communications. 16, 1, 24 p., 1703.

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