FIGURE 6.

Inhibition of nuclear export leads to accumulation of SHMT1 in the nucleus. A, HeLa cells were treated with or without 20 μm MG132 and varying levels of the nuclear export inhibitor LMB for 24 h. Nuclei were isolated from cells and lysed using SDS-PAGE loading buffer. SHMT1 immunoblot analyses were completed as described in Fig. 2. Lamin A immunoblots were performed to control for loading. B, nuclear purity was assessed by GAPDH immunoblotting. C, SHMT1 immunoprecipitations (IP) were performed using nuclear extracts from cells treated with 2.5 ng/ml LMB (lane 2) or 2.5 ng/ml LMB and 20 μm MG132 (lane 3) as described in Fig. 3. Non-immune IgG was used as a negative control for samples treated as in lane 3. D, SHMT1 immunoprecipitations were performed using nuclear extracts from cells treated with 2.5 ng/ml LMB (lane 3) or 2.5 ng/ml LMB and 20 μm MG132 (lane 2). Non-immune IgG was used as a negative control for samples treated as in lane 2. A darker exposure of this blot shows the presence of multiple higher molecular weight SHMT1-SUMO-2/3 conjugates in samples treated with MG132 (lane 3). All experiments were repeated at least three times with similar results.