Figure 6.

Expression of wild type TRPM2 but not E960D restores expression of Nrf2 and antioxidant enzymes, and Nrf2 nuclear translocation in TRPM2 depleted cells. SH-SY5Y TRPM2-KO cells were stably transfected with empty vector (KO-v), wild type TRPM2 (KO-FL), or E960D (KO-E960D). Scrambled controls were transfected with empty vector (Scr-v). Cells were treated with 0.3 µM doxorubicin for 24 hours. (a) Western blotting was performed with antibodies to TRPM2-C, Nrf2, proteins which regulate Nrf2 (IQGAP1, Keap1), antioxidant enzymes regulated by Nrf2 (PRX3, GSTP1, GSR1), SOD2, proteins involved in NADH and NADPH generation (ALDH1L2, MTHFD1, MTHFD2, MTHFR, G6PD), proteins involved in GSH synthesis (GLS, GCLM), c-Myc, and actin. Two experiments were performed and representative Western blots are shown. Intensify of bands was quantitated with Li-Cor technology. Blots were normalized by comparing bands to each protein’s average scrambled control. Normalized means ± SEM for each protein and group from two experiments are shown (n = 4). (b) Cells lysates were separated into cytoplasmic and nuclear fractions and Western blots were probed with antibodies to IQGAP1 and Nrf2. Lamin A/C (nuclear) and GAPDH (cytoplasmic and nuclear) were probed to demonstrate quality of fractionation. Representative Western blots from three experiments are shown. Mean ± SEM of densitometry measurements from the three normalized to Scr-v control is shown on the right (n = 3). For (a) and (b), *indicates p < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001, one-way ANOVA.