Figure 4.

Accumulation of PML SUMO1 conjugation in the nucleus. (a) K490 is the major SUMOylated site of PML protein in HeLa cells. HeLa cells were transfected with HA-PML4 (wild-type, K65/160R, K65/490R, K160/490R, and 3KR mutants) and the resulting WCEs were immunoprecipitated with anti-HA antibody-conjugated beads followed by immunoblotting with anti-acetyl-lysine and anti-HA antibodies. (b) The SV-40 nuclear localization sequence was introduced into HA-PML4 constructs (wild-type and K487R mutant) (upper panel). HeLa cells were transfected with NLS-HA-PML4 (wild-type and K487R mutant) and immunostained with anti-HA antibody (lower panels). (c) HeLa cells were transfected with HA-PML4 (wild-type and K487R mutant) and NLS-HA-PML4 (wild-type and K487R mutant), WCEs prepared and immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-acetyl-lysine and anti-HA antibodies. Note that NLS-HA-PML4 proteins migrate slower than HA- PML proteins due to the addition of NLS at the N-terminus. (d) SUMO1 modification on PML occurs in the nucleus. HeLa cells were transfected with NLS-PML4, PML4 (487R), or NLS-PML4 (K487R). Note that 20 μM of NEM was added in the lysis buffer in (b). The resulting WCEs were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-acetyl-lysine and anti-HA antibodies