FIGURE 2.

In vitro SUMOylation and dependence on Hsp27. A, identification of SUMO-1 modified WT and F508del NBD1. Immunoblots were performed using monoclonal anti-NBD1 (antibody 660) and polyclonal anti-SUMO-1 antibodies, detected with IRDye 680RD donkey anti-mouse or IRDye 800CW donkey anti-rabbit secondary antibodies using a Li-Cor Odyssey. B, selective SUMO-1 modification of F508del-NBD1 requires the SUMO E1 and E2-conjugating enzymes and ATP and is reversed by the SUMO protease, SENP-1. Purified 1S-NBD1, WT or F508del, was incubated with SUMOylation components for 60 min at 27 °C. The reaction mixture was resolved by SDS-PAGE, and NBD1 was detected by antibody (antibody 660). C, preferential modification of F508del-NBD1 by SUMO-2 in vitro is augmented by Hsp27. SUMOylation reactions were run with purified paralogs, and NBD1 was detected as in B. SUMOylated F508del-NBD1 densities were quantified and normalized to their respective control values from three independent experiments. *, p = 0.023; **, p = 0.008.