FIGURE 3.

Hsp27-mediated degradation and SUMOylation of F508del-NBD1 in vivo requires Lys⁴⁴⁷. A, the SUMO consensus site, Lys⁴⁴⁷, was mutated to generate CD4T-F508del-NBD1-K447R; this mutant and its control were expressed in HEK293 cells with Hsp27 or vector control, as above. After 48 h, F508del-NBD1 expression was assessed by IB using the indicated antibodies. Protein levels were quantified and normalized to the vector control from three independent experiments. **, p = 0.001; *, p < 0.05. B, HEK293 cells were transfected with the plasmids used in A. NBD1 SUMOylation was detected by NBD1 IP followed by IB with antibodies against NBD1 or endogenous SUMO-2/3. HC, antibody heavy chain. The SUMO signal was normalized to the amount of NBD1 pulled down and then normalized to control; data from three independent experiments. *, p = 0.007. C, K447R does not abrogate the impact of Hsp27 on FL F508del degradation. F508del CFTR or F508del CFTR-K447R and Hsp27 or control plasmids were transfected into HEK293 cells as above. Protein expression was detected by IB using the indicated antibodies. F508del CFTR levels in Hsp27 co-expressing cells were normalized to their respective controls from four independent experiments. **, p = 0.008.