Goat anti mouse igg

High-binding 96-well ELISA plates (Costar, Corning) were coated overnight with goat anti-mouse IgGs, anti-mouse IgMs (Jackson ImmunoResearch), or anti-mouse IgG1 antibodies (Southern Biotech) (250 ng/well in PBS). Plates were washed with 0.05% Tween-20-PBS (PBST), blocked for 2 h with 2% BSA, and 1 mM EDTA-PBST (Blocking buffer). After PBST-washings, plates were incubated for 2 h with 1:100 PBS-diluted plasma and seven consecutive 1:3 dilutions in duplicate. Purified mouse IgG, IgG1 (Sigma-Aldrich), and IgM (Merck Millipore) antibodies starting at 12 µg/ml and seven consecutive 1:3 dilutions in PBS were used as standards. After PBST-washings, plates were incubated for 1 h with HRP-conjugated goat anti-mouse IgGs, anti-mouse IgM (Jackson ImmunoResearch), or rat anti-mouse IgG1 antibodies (Southern Biotech) in blocking buffer, washed, and revealed by adding HRP chromogenic substrate (ABTS solution, Euromedex). Experiments were performed at room temperature using HydroSpeed™ microplate washer and Sunrise™ microplate absorbance reader (Magellan v7.2, Tecan Männedorf), with optical density measurements made at 405 nm (OD405 nm).

Protein G-Sepharose was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Precast NuPAGE® Bis-Tris gels were from Thermo Fisher Scientific (Waltham, MA, USA). NE and AngII were bought from MedChemExpress (Shanghai, China). All other chemicals were from Sigma-Aldrich (Shanghai, China) or Sangon Biotech (Shanghai, China). The commercial primary antibodies used in this study are listed in Supplementary Table 1, and used at a dilution of 1:1000 for immunoblotting. The RalGAPα1, RalGAPα2 and RalGAPβ antibodies were described previously^{14 (link)}, and used at a dilution of 1:1000 for immunoblotting. The antibody recognizing pThr⁴⁸⁴-SERCA2a was as previously reported^{7 (link)}, and used at 1 µg/ml for immunoblotting. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cat No. 111-035-003), mouse anti-rabbit IgG (211-002-171), goat anti-mouse IgG (Cat No. 115-035-003) and goat anti-mouse IgG (115-005-174) were from Jackson ImmunoResearch Labs, and used at a dilution of 1:5000. Plasmids for RalGAPα1, RalA and SERCA2a were reported previously^{7 (link),16 (link)}.

Crosslinking of hCD59 was achieved with anti-CD59 mAb MEM-43 (1 μg/ml, ThermoScientific, Waltham, MA, USA) and goat anti-mouse IgG (1 : 100, Jackson ImmunoResearch, West Grove, PA, USA). hCD59 crosslinking was also achieved with a GFP and His-tagged version of the VLY binding domain (10 μg/ml, VLYD4-GFP) and mouse anti-His mAb (1 : 100, Sigma-Aldrich, St. Louis, MO, USA) or chicken anti-GFP pAb (1 : 100, EMD Millipore, Billerica, MA, USA). hCD55 crosslinking was achieved with anti-CD55 mAb (10 μg/ml, ThermoScientific) and goat anti-mouse IgG (1 : 50, Jackson ImmunoResearch). As negative controls, His-tagged PLYD4 (10 μg/ml) and anti-His mAb (1 : 50, Sigma-Aldrich) or rGFP and chicken anti-GFP pAb (1 : 50, EMD Millipore) was added to RBCs. For hemolysis assays, receptor crosslinking was performed concurrently with PFT treatment. For immunoprecipitations (IPs), receptor crosslinking was performed at 37 ^oC for 30 min followed by the IP procedure.