Fig 1: shRNA knockdown of p38MAPK-α reproduces the effects of SB203580 treatment.A, differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α (one shRNA) and p38MAPK-γ isoforms (two shRNAs), incubated with doxycycline for 2 days, and subsequently lysed and Western blotted for expression of p38MAPK isoforms (using anti-p38MAPKα antibody 9212 from Cell Signaling) or phosphorylated p-p38MAPK. Cropped lanes are derived from the same membrane. B, the same cells were analyzed by Western blotting for expression of αSyn in the conditioned medium, and αSyn, LC3, and p62 in the lysate. C–E, quantitation of Western blot band absorbances from aforementioned experiment for double p38MAPK-α and p38MAPK-γ knockdown for (C) αSyn in conditioned medium, or (D) αSyn, and (E) LC3-II in the lysate fraction. (Kruskal–Wallis test, N = 4–6). F, LDH release from the same cells (ordinary one-way ANOVA, N = 4). G, differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used. Cropped lanes are derived from the same membrane. H and I, the graphs show quantitation of levels of p38MAPK-α isoform (unpaired t test, N = 4) and active (phosphorylated) p38MAPK (one-sample t test, N = 4). J, secretion of αSyn to the medium (unpaired t test, N = 3) as performed in (G). K, LDH release to the medium (unpaired t test, N = 4). All graphs show mean ± SEM. αSyn, α-synuclein; LDH, lactate dehydrogenase.