Skip to main content
. 2013 Jun 12;8(6):e65149. doi: 10.1371/journal.pone.0065149

Figure 2. Characterization of cytoplasmic SMC3 protein.

Figure 2

(A) Expression of mRNA of Smc1, Smc3 and Rad21 72 h after esiSMC1 treatment, compared to control (esiEGFP), was examined by real-time RT-PCR. Relative quantification of gene expression was achieved by normalization to ß-actin, (n = 3). (B) Effect of SMC1 knockdown on RAD21 protein in total RIPA extracts as monitored by IB with anti-Rad21 antibody 72 h post esiSMC1 or esiEGFP transfection. Bottom: quantification of IB from three independent experiments. (C) Sequential IP. IP #1 from nuclear and cytoplasmic extracts from esiSMC1-treated and control cells. The supernatant was used for IP#2 with anti-SMC3 antibody. Eluates were analyzed by IB using anti-RAD21, -SMC3, and -SMC1 antibodies. (D) Nuclear export was inhibited 70 h after esiSMC1 or control treatment by addition of LMB to a final concentration of 5 ng/mL for another 2 h. The localization of SMC3 (in red, top half) was examined by immunofluorescence. NFκB (in red, bottom half) was used to confirm the LMB effect. DNA was visualized by DAPI (in blue). (E) Immunoprecipitation of SMC3 from cytoplasm and nuclear extracts after LMB inhibition was performed and eluates examined by silver staining. Arrows indicate the positions of SMC1, SMC3 and RAD21. The asterisk indicates an unspecific band as specified by mass spectrometric analysis.