Fig. 4

a Cell extracts from NIH3T3-Smn_RNAi cells expressing SMN-WT or SMN-2VA and treated with doxycycline (Dox) for 7 days were fractionated by 10-30% sucrose gradient centrifugation. SMN in each fraction was detected by SDS-PAGE and Western analysis. Number of fractions and sedimentation (S) values are indicated. b Representative images of NIH3T3-Smn_RNAi cells expressing SMN-WT or SMN-2VA and cultured with Dox for 7 days and stained with anti-flag antibody. Scale bar, 10 μm. c Quantification of the number of cells with nuclear Gems in NIH3T3-Smn_RNAi cells expressing SMN-WT or SMN-2VA and cultured with Dox for 7 days. Data represent means and SEM of three independent experiments (n = 3). Statistical significance was determined by two-tailed unpaired t test (P value = 0.0007). d Representative western blot analysis of SMN levels in NIH3T3-Smn_RNAi cells expressing SMN-WT or SMN-2VA and treated with Dox for 7 days and cycloheximide at different time points. Tubulin is used as loading control. e Quantification of the SMN levels from three independent Western blots experiments as in (d) (n = 3 independent biological experiments). Data represent mean and SEM. f Equal amounts of cell extracts from NIH3T3-Smn_RNAi cells expressing SMN-WT or SMN-2VA and treated without (−) or with (+) Dox for 7 days were analyzed in snRNP assembly reactions by immunoprecipitation of radioactive U1 snRNA with anti-SmB (18F6) antibody followed by electrophoresis on denaturing polyacrylamide gels and autoradiography. g The amount of U1 snRNA immunoprecipitated in snRNP assembly experiments as in (f) was quantified using a Typhoon Phosphorimager and expressed as a percentage of that in samples without Dox treatment. Data represent means and SEM of three independent biological experiments (n = 3). Statistical significance was determined by two-sided, unpaired multiple t tests followed by Holm-Sidak correction (adjusted P values −Dox vs. +Dox: RNAi = 0.000011; WT = 0.020757; 2 VA0.000319).