Fig. 1

Fig. 1

a SDS-PAGE and Western blot analysis of endogenous UBC9 protein levels in HeLa wildtype (WT) or UBC9_RNAi cells cultured without (-) or with (+) Doxycycline (Dox) for 4 days. A twofold serial dilution of WT HeLa cell extract is analyzed on the left. b Western blot analysis of anti-SUMO-2/3 immunoprecipitates in HeLa WT or UBC9_RNAi cells cultured without (−) or with (+) Dox for 4 days. c Representative fluorescent images of HeLa UBC9_RNAi cells cultured without (−) or with (+) Dox for 4 days and stained with antibodies against SMN, GEMIN2, GEMIN3, GEMIN5, and GEMIN6 (green), and with DAPI (blue). Cytoplasmic foci are indicated with white arrowheads. Scale bar, 10 μm (figures are representative of n = 3 biologically independent experiments). d Quantification of number of cells with cytoplasmic foci from the same groups as in (c). Data represent means and SEM (n = 3 biologically independent experiments). Statistical significance was determined by two-sided, unpaired multiple t tests followed by Holm-Sidak correction (adjusted P values −Dox vs. +Dox: SMN = 0.023007; GEMIN2 = 0.026589; GEMIN3 = 0.010270; GEMIN5 = 0.023007; GEMIN6 = 0.023007). e Cell extracts from HeLa UBC9_RNAi cells cultured without (−) or with (+) Dox for 4 days were fractionated by 10–30% sucrose gradient centrifugation. Endogenous SMN levels in each fraction were analyzed by SDS-PAGE and Western blot. Numbers of fractions and sedimentation (S) values are indicated.