Fig 2: USP6 interacts with NMDA receptors.(A) Characterizing interactions between USP6-HA and glutamate receptor subunits by co-IP. WT and CAM-USP6 mouse brain lysates were precipitated with anti-HA magnetic beads, and precipitates were subsequently immunoblotted with antibodies to detect GluN1, GluN2B, GluA1, GluA2, APP, and β-actin. (B) co-IP interactions between exogenously expressed GluN1-Myc and Flag-tagged USP6, TBC1D3, or USP32. GluN1-Myc and USP6-Flag/TBC1D3-Flag/USP32-Flag were detected in lysates from cotransfected HEK293T cells; anti-Flag complexes were immunoprecipitated from lysates with an anti-Flag antibody and immunoblotted with a Myc antibody. (C) co-IP interactions detected between exogenously expressed GluN2B-Myc and USP6-Flag/TBC1D3-Flag/USP32-Flag. GluN1-Myc and USP6-Flag/TBC1D3-Flag/USP32-Flag were cotransfected into HEK293T cells, and complexes were immunoprecipitated from lysates with an anti-Flag antibody and immunoblotted with a Myc antibody. APP, amyloid precursor protein; CAM, CamK2a; co-IP, coimmunoprecipitation; Glu, glutamate ionotropic receptor; HA, hemagglutinin; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; NMDA, N-methyl-D-aspartate; TBC1D3, TBC1 domain family member 3; USP, ubiquitin-specific protease; WT, wild-type.

Fig 3: GORASP2 increases HMGB1 secretion. (A, B) HEK293T cells were transfected with GORASP2-MYC, shRNA control (SHC001) or shGORASP2 (#1; TRCN0000129348 and #2; TRCN0000127611). The cells were incubated in EBSS media for 14 h. WCLs were immunoblotted with anti-MYC, anti-HMGB1, anti-GORASP2, and anti-ACTB antibodies. The culture supernatants were concentrated with Amicon Centricon filters. (C) GORASP2-MYC and HSP90AA1-Flag were transfected into HEK293T cells. WCLs were immunoblotted with anti-MYC, anti-Flag, anti-HMGB1, and anti-ACTB antibodies. Culture supernatants were concentrated with Centricon filters. Results were compared to those for co-expressed HSP90AA1 and GORASP2/GRASP55. (D) GORASP2-MYC was transfected and treated with 0.5 μM GA for 24 h. WCLs were immunoblotted with anti-MYC, anti-HMGB1, and anti-ACTB antibodies. Culture supernatants were concentrated with Centricon filters. Data are presented as the mean ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey honestly significant difference posthoc test for multiple comparisons

Fig 4: (A and B) DARPins strongly exhibited the ability to neutralize (A) and bind (B) the different TcdB toxins. conc., concentration. (C) TcdB neutralization potency of different DARPins and bezlotoxumab. For neutralization assays, serially diluted immobilized-metal affinity chromatography (IMAC)-purified DARPins were mixed with the appropriate toxin and then added to Vero cells seeded the night before in 96-well plates. The cell viability was quantified by the CellTiterGlo assay 72 h later and normalized to naive Vero cells. For ELISAs, the MaxiSorp plates were coated with the appropriate toxin followed by treatment with serially diluted DARPins. The amounts of plate-bound DARPins (containing Myc tags) were quantified using an anti-c-Myc antibody. Data in panel A represent averages of results from at least 2 independent experiments. Data presented in panel B are representative of results from two independent experiments performed in duplicate.

Fig 5: DHODH inhibitors promote GLUT4 translocation to the plasma membrane and affect mitochondrial respiration and glycolysis(A) Localization of GLUT4 upon treatment with the indicated compounds. Plasma membrane-bound GLUT4 is labeled with a Myc tag on its extracellular domain, and total GLUT4 is labeled with mCherry. The average (±SEM) of the ratio between anti-Myc and mCherry fluorescence was calculated. p values correspond to Student's t test, and n = 23−30 cells for each treatment.(B and C) Cellular respiration and glycolysis measurements.(B) Average (±SEM) oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements.(C) Variation of respiration and glycolysis parameters in response to the indicated inhibitors. Values correspond to the average (±SD). n = 3 biological repeats, and p values correspond to Student's t test. See also Figure S2. +U, +uridine 100 μM; BAY, BAY2402234; brq, brequinar