Research Paper Volume 12, Issue 13 pp 13388—13399

Figure 1. CBR-470-1 activates Nrf2 signaling cascade in SH-SY5Y cells. Differentiated SH-SY5Y neuronal cells were treated with CBR-470-1 (10 μM) or the vehicle control (“Veh”), cells were further cultured for the applied time periods, Keap1 immunoprecipitation with Nrf2 was tested by a Co-IP assay (A); Nrf2 and Keap1 protein expression in total cell lysates was tested as “Inputs” (A). SH-SY5Y neuronal cells were pre-treated with MG-132 (10 μM) for 24h, followed by CBR-470-1 (10 μM) stimulation and cultured for another 4-8h, Nrf2 and Tubulin protein expression in total cell lysates was tested (B). SH-SY5Y cells were pretreated with cycloheximide (CHX, 100 μg/mL) for 12h, followed by CBR-470-1 (10 μM) stimulation and cultured for another 4h, Nrf2 and Tubulin protein expression in total cell lysates was shown (C). SH-SY5Y neuronal cells were treated with CBR-470-1 (10 μM) or the vehicle control (“Veh”), cells were further cultured for the applied time periods, expression of indicated Keap1-Nrf2 pathway genes was tested by qPCR and Western blotting analyses (D, F, G); Alternatively, cells were harvested and relative ARE luciferase activity was tested, with results normalized to that of vehicle control (E). Expression of listed proteins was quantified and normalized to the loading control (A–D, G). Data were expressed as mean ± standard deviation (SD, n=5). * P< 0.05 vs. “Veh” cells (E, F). Experiments were repeated four times with similar results obtained.