Antiphospho elf2α

To evaluate the unfolded protein response (UPR) in CDs/y and CDr/y provided RD or DD, we studied protein expression in homogenates of the pancreas in CDs/y and CDr/y after 4 days, 1 week, 2 weeks, 3 weeks, and 4 weeks from initiation of DD or RD. We measured by Western blot the expression level of GRP78/BiP and of key proteins involved in initiating UPR: the PERK (eukaryotic initiation factor 2α -EIF2α, Activating Transcription Factor 4-ATF4 and the C/EBP homologous protein–CHOP) and the IRE (X box binding protein 1-XBP-1) pathways. As shown in Figure 1, PERK activation induces EIF2α phosphorylation which increases transcription of AFT4 that increases in turn CHOP levels and IRE1α activation increases transcription of XBP-1 (and JNK which we did not study). In order to study expression of GRP78/BiP, we used as primary antibody Anti-GRP78 from Abcam ab (1:50,000) (75kDa). For Phospho eif2α/Eif2α, we used Anti-elF2α (38 kDa, 1:1000) and Anti-Phospho-elF2α (38kDa, 1:1000) from Cell Signaling technology. For ATF-4, we used Anti-ATF-4 (EPR18111) from Abcam ab184909 (50 kDa, 1:700). For CHOP, we used Anti DDIT3 (Chop) from Abcam ab179823 (25 kDa, 1:1000). For XBP1, we used Anti XBP-1S (E8Y5F) from Cell Signaling technology (50 kDa, 1:1000).