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. Author manuscript; available in PMC: 2012 Aug 1.
Published in final edited form as: Nat Cell Biol. 2012 Jan 8;14(2):192–200. doi: 10.1038/ncb2408

Figure 3.

Figure 3

LPS promotes dephosphorylation of eIF2Bε through a mechanism involving PP2A, with evidence in vivo. (a) Macrophages from wild-type or Trif-/- mice were untreated or pretreated ± LPS (1 ng ml–1) for 24 h followed by treatment with tunicamycin (TN, 1 μg ml–1) for 90 min. In an additional LPS-TN group, the phosphatase inhibitor okadaic acid (1 nM) was added at the same time as LPS. Cells extracts were then added to a solution of purified 32P-labeled eIF2Bε, and the rate of dephosphorylation was determined as described in Methods. Data are expressed as mean ± s.e.m. with n = 6; *P < 0.05 compared with Con and the corresponding Trif-/- group. **P < 0.05, compared with TN and the corresponding Trif-/- group. (b) Macrophages were transfected with scrambled RNA or Pp2ac siRNA, and 48 h later the cells were treated similarly to the first 3 wild-type groups in (a). Cell extracts were analyzed by immunoblot for p- and total eIF2Bε, CHOP, PP2Ac, and β-actin. The data were then quantified by densitometry. *P < 0.05 and **P < 0.02 vs. TN in the scrambled RNA groups. (c) MEFs were either mock-transfected or transfected with a plasmid encoding PP2Ac with a Tyr307-Phe mutation or a Leu199-Pro mutation. 12 h post-transfection, cells were then treated ± LPS (500 ng ml–1) for 8 h followed by a 2-h treatment with tunicamycin (TN; 0.5 μg ml–1). Cell extracts were subjected to immunoblot analysis for CHOP and β-actin. Densitometric quantification of the immunoblot data are shown in the graph. *P < 0.001 vs. TN group; #P < 0.03 vs. LPS-TN group; n.s., not significant. (d) Macrophages from wild-type or Trif-/- mice were untreated or pretreated ± LPS (1 ng ml–1) for 24 h followed by treatment with tunicamycin (TN, 1 μg ml–1) for 3 h. Extracts were analyzed by immunoblot for phospho (p)- and total PP2Ac and then quantified by densitometry; *P < 0.05 vs. control and TN groups for wild-type macrophages and vs. all groups for Trif-/- macrophages. (e) Mice were injected intravenously with LPS (80 μg kg-1) or vehicle control once a day for 2 consecutive days and then injected with tunicamycin (TN, 1 mg kg-1) intraperitoneally. Twelve hours later, the mice were sacrificed, and liver extracts were assayed by immunoblot for p- and total PP2Ac, p- and total eIF2Bε, and β-actin and then quantified by densitometry. *P = 0.04; **P = 0.0004; #P = 0.015 . (f) As in (e), except the mice were sacrificed 24 h after the tunicamycin injection, and kidney extracts were assayed. *P = 0.02 compared with Con; **P = 0.04 compared with TN. +P < 0.05 compared with TN; #P < 0.01 compared with TN. All densitometry data are expressed as mean ± s.e.m. with n = 3, except n = 4 for (e) and (f).