Characterization of synemin M in mouse ES cells. A, Analysis by RT-PCR of mRNA encoding synemins H and M isoforms and nestin in USP-1 (lane 1), CGR8 (lane 2) and mouse embryo at E9.5 (lane 3). Synemin M mRNA was found in pluripotent ES cells, while synemin H and nestin mRNAs were absent (lanes 1 and 2). Both synemin isoforms were detected in the 9.5-day-old embryo (lane 3). B, Characterization of synemins H, M and Oct-3/4 proteins by Western blot. USP-1 (lane 1), CGR8 (lane 2) ES cells and the adult mouse bladder (lane 3) were used for this study. Only the synemin M and Oct-3/4 were found in pluripotent ES cells (lanes 1 and 2). The synemin H was absent from undifferentiated ES cells (lanes 1 and 2). The adult mouse bladder was used for a control (lane 3). C, Analysis of synemin in mouse ES cells by 2D-PAGE and immunoblotting. Total protein extract of ES cells was separated by using pH 4-6 gradients IPG strips and 8% SDS/PAGE gels. Two spot trains of synemin M with isoelectric points (pI) 5.1-5.2 and apparent 148-150 kDa molecular weights (MW) were characterized. Analysis of the phosphorylation of synemin M revealed that two spot trains of synemin M contained phosphoserines (PSer) with isoelectric variants. Syn: synemin.