831940.fig.002a
(a)
831940.fig.002b
(b)
831940.fig.002c
(c)
Figure 2: TCTP present in the nucleus is sumoylated. Lanes 1 and 2 show the proteins in the cytoplasm (CF) and nuclear (NF) fractions probed with an anti-TCTP antibody (a). The TCTP was then immunoprecipitated from these fractions using the anti-TCTP antibodies and the precipitate was subsequently probed with an anti-SUMO antibody (b). Lanes 3 and 4 show that sumoylated TCTP is present in both cytoplasm and nucleus. In order to determine if sumoylation is critical for the transport of TCTP into the nucleus, we used a FLAG-tagged TCTP to monitor the movement of TCTP inside the cell. Cos1 cells were transfected with either FLAG-tagged wild-type TCTP (W) or FLAG-tagged mutated TCTP (M). To determine whether the FLAG-tagged TCTPs were sumoylated, an anti-FLAG monoclonal antibody was used to immunoprecipitate the FLAG-tagged TCTP from the cytoplasm and nuclear fractions of the Cos1 cells. The bound TCTPs were then eluted, separated on a 12% gel, transferred to a nitrocellulose sheet, and probed with polyclonal anti-SUMO antibodies (c). These studies confirm that the high molecular weight form of wild-type TCTP present in the nuclear fraction is sumoylated. Results presented are representative of three similar experiments.