FIGURE 2.

CMA stabilizes Nrf2 through decreasing Nrf2 ubiquitination. (a). Change of Nrf2 half‐life by LAMP2A. SN4741 cells were transfected with the Flag‐LAMP2A plasmid for 48 h and treated with 100 µg/ml CHX for the indicated times. Cells were harvested for immunoblotting with indicated antibodies. Right panel shows the quantification of the Nrf2 protein level normalized to β‐actin. (Two‐way ANOVA followed by Sidak's multiple comparisons test, mean ± SEM, n = 3 independent experiments, **p < 0.01). (b). The effect of LAMP2A on Nrf2 ubiquitination. SN4741 cells were transfected with the Flag‐LAMP2A and HA‐ubiquitin (ub) plasmids for 48 h and treated with 10 μM MG132 for another 5 h. The ubiquitination of Nrf2 was determined by immunoprecipitation of Nrf2 and subsequent immunoblot with anti‐HA antibody. Right panel shows the quantification of the HA‐ub protein level normalized to IgG. The input represents 10% of total cell lysates (Student's t test, mean ± SEM, n = 3 independent experiments, **p < 0.01). (c). The effect of LAMP2A on the interaction between Nrf2 and Keap1. SN4741 cells were transfected with the Flag‐LAMP2A plasmids for 48 h. Lysates were immunoprecipitated with IgG or anti‐Nrf2 antibodies and subsequently immunoblotted with anti‐Keap1 antibody. The input represents 10% of total cell lysates