Supplemental Material

Reference

Zhang, P., McGrath, B., Li, S., Frand, A., Zambito, F., Reinert, J., Gannon, M., Ma, K., McNaughton, K., and Cavener, D.R. (2002). The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas. Mol. Cell. Biol. 11, 3864-3874.

This article contains the following supporting material:

• Figure 1 - Rate of disappearance of p90 ATF6 in the PERK-/- cells.
  Percent cleaved p90ATF6 in mouse embryonic fibroblast (MEFs) derived from perk knockout mouse (PERK-/-) treated with DTT (black solid line), thapsigargin (black dashed line), and tunicamycin (gray solid line) were shown.

• Figure 2 - PERK is the only kinase responsible for eIF2α phosphorylation during the UPR.
  (A)Immunoblots of phosphorylated eIF2α (p-eIF2α) and β-Actin from lysates of mouse embryonic fibroblast (MEFs) derived from either the wild type (Perk+/+) or perk knockout mouse (Perk-/-) (Zhang et al., 2002). Either wild type or perk knockout cells were treated with DTT, thapsigargin (Tg), and tunicamycin (Tm) for the indicated amounts of time.
  (B) Quantitation of the increase in p-eIF2α levels over the time course shown in (A). The levels of p-eIF2α were quantitated and normalized with levels of β-Actin. Fold induction of p-eIF2α in Perk+/+ (solid) or Perk-/- (open) MEFs was calculated by taking the ratio between the levels of normalized p-eIF2αƒnat time zero and each time point. The graphs represent three independent time course experiments carried out with DTT (black solid line) Tg (black dashed line), Tm (gray solid line).

• Figure 3 - Total level of eIF2α does not change during UPR time courses.
  (A)Immunoblots of total eIF2α and β-Actin from lysates of CHO cells treated with DTT, thapsigargin (Tg), and tunicamycin (Tm) for the indicated amounts of time.
  (B)Quantitation of total eIF2α levels over the time course shown in (A). The levels of eIF2α were quantitated with a Typhoon 9400 phosphorimager and normalized with levels of β-Actin. Fold induction was calculated by taking the ratio between the levels of normalized eIF2α at time zero and each time point. The graph represents three independent time course experiments carried out with DTT (black solid line), thapsigargin (black dashed line), and tunicamycin (gray solid line). Untreated is represented as a solid black line with open circles.
  (C)Immunoblots of total eIF2α and β-Actin from lysates of NIH 3T3 cells treated with DTT, thapsigargin (Tg), and tunicamycin (Tm) for the indicated amounts of time.
  (D)Quantitation of total eIF2α levels over the time course shown in (C) was carried out and plotted as described in (B).

• Figure 4 - Production of ATF4 protein during ER stress correlates with phosphorylation of eIF2α in CHO cells.
  (A)Immunoblots of ATF4 from lysates of CHO cells treated with DTT and thapsigargin (Tg) for the indicated amount of time are shown.
  (B)Quantitation of ATF4 levels over the time course shown in (A). DTT is represented as a black solid line, and thapsigargin as a black dashed line.