Fig. 2
ER stress does not mediate JNK activation in differentiated PC12 or SH-SY5Y nerve cells. a–d PC12 cells expressing α-SNC/p25α were transduced with control vector or dominant-negative ASK1-K709R (ASK1-DN) with a viral dose of 25 or 50 μl. a Two days post-infection cells transduced with control or 50 μl ASK1-DN vector were prepared for immunofluorescence with anti-ASK1 antibodies. Bar, 10 μm. b PC12 α-SNC/p25α cells transduced with control or ASK1-DN vector were stained with anti-ASK1 antibodies and analyzed by flow cytometry. Results are expressed as mean fluorescent intensity (MFI) of ASK1 staining (mean and SEM, N = 2). c Cell lysates prepared from transduced PC12 cells were western blotted using anti-p-JNK antibodies. d Densitometric quantitation of p-JNK western bands from above. Results are expressed as mean integrated optical density (IOD) normalized to control-transduced cells (mean and SEM, N = 4). e, f ATRA-differentiated SHSY-5Y cells expressing α-SNCA30P alone or together with p25α were treated overnight with tunicamycin (Tun; 700 nM), thapsigargin (Thaps; 25 nM), or bafilomycin A (Baf; 20 nM). e Cell lysates were then western blotted with anti-CHOP, anti-LC3B, and anti-p-JNK antibodies. All lanes shown for each respective antibody were derived from the same membrane. f Densitometric quantitation of p-JNK western bands from above. Results are expressed as mean and SEM integrated optical density (IOD) normalized to (respective) control-transduced cells (N = 3, *p < 0.05 by Student t test compared to control)