Figure 6

Nuclear entry of SHPR 190 is energy-independent. (a) Jurkat cells were cultured either in RPMI 1640 plus FCS at 37°C (lane 1) or 4°C (lane 2), or in HBS buffer containing 10 mM glucose (ctl, lane 3) or 10 mM deoxyglucose plus 1 μM rotenone (rot, lane 4) or 10 mM glucose plus 5 mM amiloride (ami, lane 5) for 30 min. Cells were then incubated with 2 μM of SHPR190 for 2 h, washed in HBS and lysed in RIPA buffer. Cell extracts were analysed by immunoblot using a monoclonal antibody to SUMO-1 (top panel) and to β-actin (bottom panel). (b) and (c) Jurkat cells were incubated for 2 h either with 4 μM of FP1UG or with 2 μM of SHPR190 in HBS buffer at 37°C. Cells were then washed with HBS and fractionated into cytoplasmic (c, lane 1) nuclear (n, lane 2), and membrane fractions (m, lane 3). Samples of each fraction were analysed by immunoblot using anti-GFP (B) or anti SUMO-1 (C) antibodies (top panels). Cell fractionation was controlled by immunoblot using anti-RRM2 (ribonucleotide reductase peptide M2) antibody (bottom panels). (d) Jurkat cells were cultured in HBS buffer either at 37°C (lanes 1 to 3) or 4°C (lanes 4 to 6), or in the presence of 5 mM amiloride (lanes 7 to 9) 30 min before addition of 2 μM of SHPR190. 2 h after incubation, cells were washed in HBS and fractionated into cytoplasmic (m), nuclear (n), and membrane fractions (m). Samples from each fraction were analysed by immunoblot using the monoclonal antibody to SUMO-1.