FIGURE 1.

CMA increases the level of Nrf2 protein and its transcriptional activity. (a). The effect of LAMP2A on endogenous Nrf2 protein level. SN4741 cells were transfected with Flag‐LAMP2A plasmids for 48 h. The cell lysates were analyzed by immunoblot with the indicated antibodies. Right panel shows the relative Nrf2 level normalized to β‐actin (Student's t test, mean ± SEM, n = 3 independent experiments, **p < 0.01). (b). The effect of LAMP2A on the level of Nrf2 in the nucleus. SN4741 cells were treated as in (a). The nuclear or cytoplasmic fractions were prepared for analysis. Right panel shows the relative Nrf2 level normalized to β‐actin (Student's t test, mean ± SEM, n = 3 independent experiments, **p < 0.01). (c). The effect of LAMP2A on the expression of Nrf2 target genes. SN4741 cells were transfected with Flag‐LAMP2A for 48 h. mRNA was isolated for qPCR. The mRNA level was normalized to that of β‐actin (Student's t test, mean ± SEM, n = 6 independent experiments, *p < 0.05, **p < 0.01). (d). The effect of serum deprivation on endogenous Nrf2 protein level. SN4741 cells were maintained in serum‐free media for 48 h. The cell lysates were analyzed by immunoblot with the indicated antibodies. Right panel shows the relative Nrf2 level normalized to β‐actin (Student's t test, mean ± SEM, n = 3 independent experiments, **p < 0.01). (e). The effect of serum deprivation on the level of Nrf2 in the nucleus. SN4741 cells were treated as in (d). The nuclear or cytoplasmic fractions were prepared for analysis. Right panel shows the relative Nrf2 level normalized to β‐actin (Student's t test, mean ± SEM, n = 3 independent experiments, **p < 0.01). (f). The effect of serum deprivation on the expression of Nrf2 target genes. SN4741 cells were treated as in (d), mRNA was isolated for qPCR. The mRNA level was normalized to that of β‐actin (Student's t test, mean ± SEM, n = 6 independent experiments, *p < 0.05)