Figure 5. The mutant γ2(Q390X) subunit protein increased conjugation with ER chaperones and caused ER stress.
(A-B) Total lysates from HEK293T cells expressing γ2FLAG subunits (cDNA 3μg) were immunoprecipitated with FLAG beads and immunoblotted with anti-FLAG antibody (A) or GRP78 (B, upper panel) or calnexin (B, lower panel) antibody. The γ2FLAG subunits, GRP78, and calnexin were normalized to the wildtype condition. In A, the γ2FLAG subunits were measured from about 75 KDa to 250 KDa. Individual wildtype γ2FLAG subunits were predicted to be 55 KDa and migrated at about 50 KDa, and individual FLAG-tagged truncated mutant γ2(Q390X) subunits migrated at lower molecular masses predicted to be about 40 KDa. (C). Total lysates from mouse L929 cells expressing wildtype or mutant γ2 subunits in combination with the wildtype partnering α1 and β2 subunits were analyzed by SDS-PAGE and immunoblotted with mouse monoclonal anti-GADD153 antibody. In A-D, LC stands for loading control GAPDH. (D). The nuclei portion of the total forebrain of Gabrg2+/Q390X knockin mice at different ages was purified and lysed. Equal amounts of the protein were analyzed by SDS-PAGE and immunoblotted with GADD153. (E-F). Total mutant subunit band IDVs of γ2FLAG subunits (E) or the conjugated chaperone GRP78 and calnexin (F) were normalized to the wildtype conditions. (G) The total amounts of endogenous GADD153 were normalized to untreated controls (con). (H) The endogenous GADD153 in the nuclei of forebrain in the Gabrg2+/Q390X knockin mice at different ages were normalized to wt in the youngest group (<1 month). Seizure onset is around P19 while mutant protein aggregates become detectable around 6mo. (In E, F and G, *p < 0.05, **p < 0.01, ***p<0.001 vs wt. In G, §§§ P< 0.001 vs con, †† vs P< 0.01 vs con+Tuni, N=5 batches of cells. In H, §§§ P< 0.001 vs 1 month old. In E to G, n=5 batch of cells, in H, N=4 mice for each group.)