Fig. 6: SNX25 activated Ngf production by inhibiting ubiquitin-mediated degradation of Nrf2.
a, Ngf mRNA expression in BMDMs transfected with either Nrf2 siRNA or scramble siRNA analyzed semi-quantitatively by RT–qPCR (siNrf2, n = 4; siCtr, n = 4, P = 0.01). b, Representative immunoblot showing Nrf2 protein levels in 293T cells in the presence or absence of MG132. Arrow, Nrf2 (61–68 kDa); arrowhead, poly-ubiquitinated Nrf2 (100–110 kDa). c, Ubiquitination levels of Nrf2 protein in 293T cells transfected with Snx25 siRNA or scramble siRNA in the presence of MG132. Arrow, Nrf2; arrowhead, poly-ubiquitinated Nrf2. The band intensity of poly-ubiquitinated Nrf2 (Ub-Nrf2) was analyzed semi-quantitatively (siCtr, n = 3; siSnx25, n = 3, P = 0.017). d, Representative immunoblot showing Nrf2 and poly-ubiquitinated Nrf2 levels in 293T cells transfected with the full-length Snx25 expression vector (Snx25 OE) or empty vector (EV) in addition to the Nrf2 expression vector in the presence of MG132. Arrow, Nrf2; arrowhead, poly-ubiquitinated Nrf2. Semi-quantitative analysis of poly-ubiquitinated Nrf2 bands is shown (empty vector and Nrf2 vector, n = 8; Snx25 vector and Nrf2 vector, n = 8, P = 0.006). e, Immunoblot of Nrf2 in BMDMs of Snx25Cx3cr1-cKO mice treated with 4-OHT or vehicle in the presence of MG132 (4-OHT, n = 7; vehicle, n = 7, P = 0.093). Arrow, Nrf2; arrowhead, poly-ubiquitinated Nrf2. f, Co-immunoprecipitation (IP) of SNX25 and Nrf2 in 293T cells expressing Snx25 and Nrf2. Cell lysates were immunoprecipitated with anti-Nrf2 antibody and immunoblotted with anti-SNX25 antibody. Normal IgG (IgG) was used as a negative control. Arrow, SNX25; IB, immunoblot. g, Detection of ubiquitin-bound Nrf2 in SNX25-knockdown or scramble siRNA-treated BMDMs treated with MG132 followed by immunoprecipitation of cell lysates with anti-Nrf2 antibody and immunoblotting with anti-ubiquitin antibody. h, Representative immunoblot of HO-1 in the hind paw skin of WT and Snx25+/− mice. i, Ngf mRNA quantification by RT–qPCR in BMDMs transfected with either Keap1 siRNA or scramble siRNA (siCtr, n = 3; siKeap1, n = 3). Snx25+/−, P = 0.046. Results are represented as mean ± s.e.m. Statistical analyses were performed using two-tailed Welch’s t-test (a,c–e) or one-way ANOVA (i), and significant differences between group means were identified with the Tukey–Kramer test. *P < 0.05, **P < 0.01.
