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. Author manuscript; available in PMC: 2013 Aug 24.
Published in final edited form as: Mol Cell. 2012 Jul 5;47(4):656–663. doi: 10.1016/j.molcel.2012.06.006

Figure 4. Effect of LDLox on GAIT target gene expression and schematic.

Figure 4

(A) LDLox induces S-nitrosylation of GAPDH at Cys247. Mouse BMDM were transfected with plasmids encoding wild-type HA-GAPDH, or C152S, C156S, or C247S mutants. Cells were treated with IFN-γ and LDLox for 24 hr. S-nitrosylation of transfected GAPDH was detected in lysates by immunoprecipitation with anti-HA tag antibody followed by biotin-switch analysis. Total transfected HA-GAPDH was determined by immunoprecipitation with anti-HA tag antibody followed by immunoblot analysis with anti-GAPDH antibody. Total GAPDH and β-actin were determined by immunoblot analysis.

(B) Expression of GAPDH C247S mutant restores translational silencing of VEGF-A. Cells were transfected with plasmid encoding HA-tagged C247S mutant GADPH and treated with IFN-γ or IFN-γ plus LDLox for up to 24 hr. Expression of HA-GAPDH, L13a, VEGF-A, and β-actin was detected by immunoblot analysis.

(C) LDLox and LPS Induce GAPDH S-nitrosylation at different sites. Human PBM were transfected with plasmids encoding wild-type HA-GAPDH, or C152S, C156S, or C247S mutants. S-nitrosylation of transfected GAPDH in presence of IFN-γ plus LDLox or IFN-γ plus LPS (10 μg/ml) was detected by biotin-switch. Transfected HA-GAPDH and GAPDH-bound L13a were determined by immunoprecipitation with anti-HA tag antibody followed by immunoblot analysis with anti-GAPDH and anti-L13a antibodies. Total GAPDH and β-actin were determined by immunoblot analysis.

(D) Three-dimensional structure of GAPDH. Ribbon diagram of tetrameric GAPDH structure with individual subunits shown in different colors. Cys residues are shown in spacefill with sulfur atoms highlighted (yellow). NAD+ in active site is shown (stick model).

(E) Schematic of GAIT pathway activation and its disruption by LDLox. IFN-γ induces GAIT complex assembly that causes translational repression of inflammation-related, GAIT target genes (upper pathway). LDLox induces GAPDH S-nitrosylation and consequent L13a degradation, causing GAIT pathway dysregulation and prolonging GAIT target gene expression (lower pathway).

See also Figure S1.