(A) Yeast two-hybrid analysis with two GAPDH constructs (amino acids 1-150 or 151-333) and a rat hippocampal cDNA library. The 1-150 amino acid fragment binds with GOSPEL, but not with Siah.
(B) Tissue distribution of GOSPEL mRNA.
(C) Tissue distribution of GOSPEL protein detected with an immunopurified polyclonal antibody against GOSPEL.
(D) GOSPEL in brain tissues. In situ hybridization reveals high expression of GOSPEL in Purkinje cells of the cerebellum as well as CA1-3 pyramidal and dentate granule neurons in the hippocampus.
(E) Cellular distribution of GOSPEL. Exogenous GOSPEL in HEK293 cells (GOSPEL-HA detected by anti-HA antibody) and endogenous GOSPEL in PC12 cells (detected by anti-GOSPEL antibody) are exclusively cytosolic. Scale bar, 40 μm.

(A) Schematic representation of the binding domain of GOSPEL for GAPDH. Amino acids 160-200 GOSPEL are responsible for binding to GAPDH.
(B) Schematic representation of the interaction domain of GAPDH for GOSPEL. Amino acids 80-120 GAPDH are responsible for binding with GOSPEL.
(C) GAPDH-GOSPEL interaction with or without NMDA exposure is diminished in primary cortical neurons from nNOS deleted mice.
(D) S-nitrosylation of GOSPEL (SNO-GOSPEL) in primary cortical neurons elicited by NMDA exposure. SNO-GOSPEL was detected by biotin switch assay as an ascorbate-sensitive signal. Asc, ascorbate.
(E) Time course for S-nitrosylation of GOSPEL and GAPDH following NMDA treatment in primary granule neuron over 16 h. GOSPEL is S-nitrosylated earlier than GAPDH. S-nitrosylation of GAPDH and GOSPEL were detected by biotin switch assay.
(F) Requirement of cysteine-47 (C47) for S-nitrosylation of GOSPEL. HEK293 cells were transfected with either HA-GOSPEL or HA-GOSPEL-C47S mutant. Cells were then treated with GSH or GSNO for 16 h and subjected to biotin switch assay. The C47S mutant of GOSPEL is not S-nitrosylated.
(G) Requirement of cysteine-47 (C47) for efficient binding of GOSPEL and GAPDH. HEK293 cells were transfected with myc-GAPDH along with HA-GOSPEL or HA-GOSPEL-C47S, and treated with either GSH or GSNO for 16 h. The protein interaction was examined by co-immunoprecipitation.

(A) Inhibition of binding between S-nitrosylated GAPDH (SNOGAPDH) and Siah by the addition of purified GOSPEL in a concentration-dependent manner.
(B) Inhibition of binding between S-nitrosylated GAPDH (SNOGAPDH) and S-nitrosylated GOSPEL (SNO-GOSPEL) by the addition of purified Siah in a concentration-dependent manner.
(C) Decreased GAPDH-Siah interaction in the presence of GOSPEL. HEK293 cells were transfected with HA-GOSPEL and/or myc-Siah constructs and then treated with GSH or GSNO for 16 h. Cell lysates were co-immunoprecipitated by an anti-myc antibody, and endogenous GAPDH was detected by anti-GAPDH antibody.
(D) Contemporaneous measurement of binding between GAPDH and GOSPEL as well as GAPDH and Siah in primary neurons 16 h following NMDA treatment.

(A) GOSPEL depletion augments GAPDH-Siah binding in primary neuronal cultures after NMDA exposure. Neuron extracts were immunoprecipitated with anti-Siah antibody, and immunoprecipitates were analyzed with SDS-PAGE followed by Western blotting with anti-GAPDH antibody.
(B) Blockade of GAPDH nuclear translocation by GOSPEL. HEK293 cells were transfected with mock or HA-GOSPEL constructs followed by treatment with 200 μM GSNO for 16 h, and immunostained with anti-HA (GOSPEL) and anti-GAPDH antibodies. Representative confocal images are shown. Red, GAPDH; green, HA (GOSPEL); blue, DAPI for nucleus. Scale bar, 40 μm.
(C) GOSPEL depletion enhances nuclear GAPDH levels in primary neuronal cultures after NMDA exposure. Neurons were separated into cytosolic and nuclear fractions. Nuclear GAPDH levels were increased in GOSPEL RNAi treated cells. Anti-histone H2B and anti-actin antibodies were used as nuclear and cytosolic markers, respectively.

(A) Overexpression of GOSPEL protects neurons from excitotoxicity in a GAPDH-GOSPEL interaction dependent fashion. Primary cerebellar granule neurons were transfected with or without various GOSPEL constructs (wild-type, ΔN160, ΔN200, and C47S). Cell viability was measured 24 h after transient exposure to 300 μM of NMDA. *p<0.01, n=3, one-way ANOVA, mean ± S.E.M.
(B) Depletion of GOSPEL enhances NMDA-mediated cytotoxicity of cortical neurons. Transfection of GOSPEL RNAi in primary cortical neurons elicits a high level of neurotoxicity 12-24 h after treatment with NMDA. *p <0.01, n=3, one-way ANOVA, mean ± S.E.M. Scale bar, 20 μm.
(C) Depletion of GOSPEL enhances NMDA-mediated cytotoxicity of cerebellar granule neurons. Transfection of GOSPEL RNAi in primary granule neurons elicits a high level of neurotoxicity 12-24 h after treatment with NMDA. *p <0.01, n=3, one-way ANOVA, mean ± S.E.M.

(A) Lentivirus-mediated overexpression of GOSPEL and GOSPEL-ΔN200 in the right cortex of C57BL/6 mouse brain. Proteins are expressed at similar levels for the same amount of viral particles.
(B) Lentiviral vectors encoding either for HA-GOSPEL or HA-GOSPEL-ΔN200 were stereotaxically injected in the right cortex of mice. Sections were immunostained with a polyclonal anti-HA antibody, revealing a significant overexpression of GOSPEL and GOSPEL-ΔN200 in the injected hemisphere. Scale bar, 100 μm.
(C) Representative micrographs of mouse brain sections injected with NMDA into the right cortex at the site of viral particle injection. Brains were stained with cresyl violet. Lesions are indicated by dotted lines.
(D) Quantitative analysis of NMDA-elicited brain damage. Lesion volume was 31 ± 4% smaller in mice treated with GOSPEL than GOSPEL-ΔN200. Lesion volumes are reported as mean ± S.E.M.