Figure 5

Loss of Puma does not alter the activation of UPR markers. Dopaminergic cultures derived from Puma +/+ and -/- animals were treated with 20 μM 6-OHDA. A) Total RNA was isolated at the indicated times and ATF3 and GAPDH RNA levels were analyzed by qPCR. ATF3 levels were normalized to GAPDH levels. B) Cell lysates were collected in RIPA buffer at the indicated times and protein levels were analyzed by western blotting for ATF3 and actin. C) Western blots in B were quantitated in ImageQuant. Levels of ATF3 were normalized to actin levels. Data were analyzed by two-way ANOVA (treatment: **, p < 0.01; genotype: ns). D) Total RNA was isolated at the indicated times and Xbp-1 RNA levels were analyzed by semi-quantitative RT-PCR. Data were analyzed by two-way ANOVA (treatment: ***, p < 0.001; genotype: ns). E) Total RNA was isolated 9 hours after treatment and RNA levels of the indicated genes were analyzed by qPCR. Data are shown as fold change over untreated control and were analyzed by two-way ANOVA. CHOP (treatment: ***, p < 0.001; genotype: ns), BiP (treatment***, p < 0.001; genotype: ns), GADD34 (treatment: **, p < 0.01; genotype: ns), p58IPK (treatment: **, p < 0.01; genotype: ns). F) Cells were fixed 24 hours after treatment and stained for TH and CHOP. G) TH-positive and CHOP-positive cells were counted and the percentage of TH-positive cells that were also CHOP-positive was calculated. Data were analyzed by two-way ANOVA (treatment: ***, p < 0.001; genotype: ns).