Figure 3

TNF-induced ER stress responses in MN9D dopaminergic (DA) cells were attenuated by SMase inhibitors. A, Diff-MN9D cells were pre-treated with 5 μM Desipramine (Des) or 10 μM GW4869 followed by treatment with 5 ng/mL TNF for 24 hours. Cell lysates were harvested for SDS-PAGE and were analyzed by immunoblot using antibodies against the ER stress proteins ATF6, IRE1, and PERK or GAPDH for normalization. Dihydro-ceramide (DH-C2-Cer at 10 μM) and tunicamycin (Tunicam. at 0.1 μg/mL) were used as negative and positive controls, respectively. B, Quantification of western blots in A. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test; * denotes p < 0.05, *** denotes p < 0.001 compared to TNF alone.