Figure 1

SUMOylation of PIM1 in vitro and in cultured cells. (a) In vitro transcribed and translated ³⁵S-methionine labeled PIM1 was incubated with recombinant SAE1/2, UBC9 with SUMO1 or SUMO2 in the presence of ATP-regeneration system. SP100 was used as a positive control in the SUMOylation reaction. SUMOylation of radiolabelled PIM1 was visualized on a Phosphorimager. (b) Bacterially expressed and purified GST-PIM1 was incubated in the presence of ATP, recombinant SAE1/2, UBC9 with SUMO1 (left) or SUMO2 (right). SUMOylated PIM1 was detected by western blotting using a GST-tag antibody. (c) COS7 cells were transfected with plasmids encoding MYC-tagged PIM1 alone or in combination with 6His-SUMO1, 2 or 3 and SUMOylation assay was carried out using Ni²⁺-NTA beads, 42–48 hours post transfection. PIM1 SUMOylation was analyzed by western blotting of Ni²⁺-NTA pull-down samples using MYC-tag (9E10) antibody. Total levels of PIM1 expressed under each transfection condition were analyzed by western blotting of Input samples using MYC-tag (9E10) antibody. (d) H1299 cells were transfected with a plasmid expressing 6His-PIM1, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using PIM1 (12H8) and SUMO2 antibody to detect SUMOylated PIM1. (e) COS7 cells were transfected with plasmids expressing PIM1 alone, or with 6His-SUMO2 in combination with catalytically active Flag-SENP1 (WT) or inactive Flag-SENP1 (MT), and SUMOylation assay was performed to isolate SUMOylated proteins. PIM1 SUMOylation was analyzed by western blotting using PIM1 (12H8) antibody. Western blotting of whole cell lysate or input was done using Flag-tag antibody to confirm expression of SENP1. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. (f) Lysates from K562 cells (treated with 20 μM MG132 for 6 hours) were incubated at 30 °C for 30 minutes in the absence or presence of 50 nM recombinant catalytic domain of SUMO protease, SENP1, followed by western blotting using PIM1 (12H8) antibody.