FIG. 8.

Keap1-dependent ubiquitination of Nrf2 requires C273. (A) MDA-MB-231 cells were cotransfected with an expression vector for HA-ubiquitin (HA-Ub) (lanes 1 to 10), a Gal4-Neh2 fusion protein (lanes 3 to 10), or expression vectors for either wild-type (WT) Keap1 (lanes 5 and 6), Keap1-C273S (lanes 7 and 8), or Keap1-C151S (lanes 9 and 10). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Cells were lysed in 2% SDS and 10 mM NEM to block ubiquitin hydrolase activity. Anti-Gal4 (α-Gal) immunoprecipitates (IP) were analyzed by immunoblot with anti-HA (α-HA) antibodies. IgG, immunoglobulin G. (B) MDA-MB-231 cells were transfected with expression plasmids and treated with MG132 as described for panel A, except that no HA-ubiquitin plasmids were transfected. Total cell lysates were collected in sample buffer and subjected to immunoblot analysis with anti-Gal antibodies. (C) MDA-MB-231 cells were cotransfected with expression vectors for HA-ubiquitin and Nrf2 (lanes 1 to 6), and expression vectors for either wild-type Keap1 (lanes 1 and 2), Keap1-C273S (lanes 3 and 4), or Keap1-C151S (lanes 5 and 6). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Cells were lysed in 2% SDS and 10 mM NEM to block ubiquitin hydrolase activity. Anti-Nrf2 (α-Nrf2) immunoprecipitates were analyzed by immunoblot with anti-HA antibodies. (D) MDA-MB-231 cells were cotransfected with an expression vector for HA-Nrf2 (lanes 1 to 6) and for either wild-type Keap1 (lanes 1 and 2), Keap1-C273S (lanes 3 and 4), or Keap1-C151S (lanes 5 and 6). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Total cell lysates were collected in sample buffer and subjected to immunoblot analysis with anti-HA antibodies.