FIG 2.

Agonist-bound GR is modified by SUMO-2/3 in response to 15d-PGJ₂ exposure. (A) HEK293 cells stably expressing wtGR or GR3KR were grown in the presence or absence of 100 nM dex for 2 h and were subsequently exposed to biotinylated 15d-PGJ₂ (10 μM) for 2 h as indicated. Cells were lysed, and the biotin–15d-PGJ₂-bound proteins were subjected to affinity isolation with streptavidin beads. Formation of 15d-PGJ₂ adducts was analyzed by immunoblotting with anti-GR antibody. (B) The cells were grown as described for panel A but were exposed to 15d-PGJ₂. After 1 h, cells were lysed in a denaturing buffer and portions of lysates (20% of immunoprecipitation [IP] inputs) were analyzed by immunoblotting with anti-GR and anti-GAPDH antibodies or were subjected to immunoprecipitation with anti-GR antibody. The precipitates were analyzed by immunoblotting with anti-SUMO-1 (S1) and anti-SUMO-2/3 (S2/3) antibodies as indicated. The arrowhead and bracket indicate the position of SUMO-2/3-modified forms of GR.