Fig 2: BMP4 detection in the heart and tibia in the wild‐type (WT) mice and in the mice with chronic heart failure (Tgαq*44). Representative immunoblot demonstrating detection of BMP4 protein expression with anti‐BMP4 antibody (Abcam, Cat#ab39973, RRID: AB_2063523) in protein extracts derived from the heart of the WT‐Sed, WT‐Tre, Tgαq*44‐Sed, and Tgαq*44‐Tre mice (panel a) and Ponceau‐S staining of the same membrane demonstrating total protein loaded and bands outlined in violet for normalization of the signal shown in panel a (panel b). Representative immunoblot demonstrating detection of BMP4 protein expression with anti‐BMP4 antibody in protein extracts derived from the tibia of the WT‐Sed, WT‐Tre, Tgαq*44‐Sed, and Tgαq*44‐Tre mice (panel c) and detection of loading control (beta Tubulin, Cat#abE7, RRID: AB_2315513) on the same membrane performed after BMP4 detection (panel d). Protein ladder is a visible Precision Plus Protein Dual Color Standards (Biorad, Cat#1610374). Internal standard in the panel (a) is mouse heart muscle sample and in the panel (c) is mouse tibia sample. WT‐Sed, wild‐type sedentary mice; WT‐Tre, wild‐type trained mice; Tgαq*44‐Sed, Tgαq*44 sedentary mice; Tgαq*44‐Tre, Tgαq*44 trained mice

Fig 3: Binding, neutralization reactivity, and protein specificity of anti-CVV MAbs.A) Binding of anti-CVV MAbs to purified virus was determined by indirect (blue) and capture (black) ELISA. B) EC50 and IC50 values (shown as MAb concentrations of ng/mL) in ELISA and PRNT were determined by non-linear fit analysis, with the bottom of the curve constrained to 0 for both analyses and the top of the curve constrained to 100 for neutralization analysis, using GraphPad Prism software V6. Dark blue: EC50 values <100 ng/mL. Light blue: EC50 values 100–10,000 ng/mL. > indicates no detectable reactivity at 10,000 ng/mL. C) Western blot of reduced purified CVV with anti-CVV MAbs was prepared as described in Materials and Methods. Lane nomenclature corresponds to MAb nomenclature. D) Western blot of non-reduced purified CVV with MAb CVV16, P (positive control (CVV MHIAF)), M (Bio-Rad Precision Plus Protein Dual Color Standard (Cat# 1610374)), Gc (glycoprotein C), Gn (glycoprotein N) N (nucleoprotein), kDa (Kilodaltons).

Fig 4: Following purification of the recombinant AAEL002675 protein. Proteins were resolved via SDS-PAGE and probed for the presence of a V5 tag on the protein of interest. Recombinant AAEL002675 (43 kDa) was detected in the first elution fraction from the protein purification process. Lane 1: Precision Plus Protein Dual Color Standards™ (Bio-Rad 1610374); lane 2: S2 supernatant HisPur column flow-through; lane 3: elution fraction 3; lane 4: elution fraction 2; lane 5: elution fraction 1.

Fig 5: T387P decreases hEAAT1 transporter density in the surface membrane. (a) Representative confocal images from HEK293T cells expressing WT (left) or T387P (right) hEAAT1-YFP fusion proteins. (b) YFP-fluorescence distributions (grey values, a. u.) from WT (grey circles, black line, n = 72) and mutant (red squares, red line, n = 24) YFP-fusion proteins from HEK293T cells. Linear fits to the values point to slightly different surface insertion ratios for WT and mutant EAAT1 (slopesWT/T387P: 0.84/0.71; R 2 WT/T387P: 0.97/0.91) (c) Representative cropped region of a SDS-PAGE showing YFP-fluorescence from total lysate (lys) or surface biotinylated protein (surf) for WT and T387P hEAAT1-YFP. MWL = Molecular Weight Ladder, BioRad-Precision plus, Dual color, #1610374. The corresponding full-length gel is provided as Supplementary Fig. 3. (d) Pooled total YFP- fluorescence emissions from WT (circles) and T387P hEAAT1-YFP (squares, n = 5/5). (e) Statistical analysis of surface biotinylation from experiments as shown in c indicates a lower ratio for surface expression of expressed protein for the mutant (P t = 0.011, n = 5/5). (f) Surface insertion probability for core- and complex-glycosylated WT (black bars) and T387P (grey bars) protein.