Reduced expressions of SMN and Gemin2 in SMN-deficient ES cells. Control (C) and SMA (S) ES cells were separated from feeding fibroblasts and allowed to differentiate in vitro in the presence of RA and SAG for 5 days. Both undifferentiated and differentiated cells were harvested and lysates were prepared. Equal amounts of total proteins were separated by SDS-PAGE, and SMN and Gemin2 were detected by western blotting. The same blot was stripped and re-probed with ERK antibodies for protein loading control (A). Signals were quantified, relative ratios of SMN or Gemin2 to ERK were calculated, and the mean value ± SD of three independent experiments is presented here. Statistical analyses (Student's t test) indicate that SMA cells have significantly reduced levels of SMN and Gemin2 expression (approximately 20% of the control for SMN and 40-50% for Gemin2) (** p < 0.01 and * p < 0.05) (B). Undiff. = undifferentiated cells and Diff. = differentiated cells.