Secondary anti mouse antibody

After PMCA, we digested all samples using proteinase K (PK) at a concentration of 50 μg/mL for 1 h at 37°C. We stopped PK digestion by boiling the sample at 100°C for 10 min after mixing with NuPage or Novex sample loading buffer (NuPage Bis-Tris gels with MES buffer and Novex Tris-Glycine gels with Tris-SDS buffers). We transferred proteins onto nitrocellulose membranes (0.45 μm; Amersham Biosciences, https://www.gelifesciences.com) and probed them with monoclonal antibody 6D11 (1:20,000) for 1 h at room temperature, while we used secondary anti–mouse antibody (Sigma, https://www.sigmaaldrich.com) at 1:3,000 dilution and incubated for 1 h. We used ECL chemioluminiscent reagent (Amersham) and a Chemidoc imaging system (BioRad, https://www.bio-rad.com) to develop and capture the images.

Total protein from plant samples was extracted and separated by 12% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), as reported previously. After transfer onto nitrocellulose (Amersham, Uppsala, Sweden) by wet electroblotting, the proteins were detected with primary antibody to GFP or PVX and secondary anti‐mouse antibody (Sigma‐Aldrich, St Louis, USA). The antigen–antibody complexes were visualized using nitrotetrazolium blue chloride/ 5‐bromo‐4‐chloro‐3‐indolyl phosphate (NBT/BCIP) buffer (Sigma) under standard conditions.

Anti-GST (Cell Signaling Technology, Catalog #2622), anti-His (Cell Signaling Technology, Catalog #2366), anti-Flag M2 (Sigma, Catalog: F3165). Anti-BV envelope gp64 PE antibody (eBioscience, Catalog: 12-6991-80). Secondary anti-rabbit antibody (Sigma Aldrich, Catalog #A6154) and secondary anti-mouse antibody (Sigma Aldrich, Catalog #A4416). The primary antibodies were used in 1:1000 dilution. The secondary antibodies were used in 1:5000 dilution. Glutathione-Sepharose 4B and Ni-NTA Agarose were from Amersham Pharmacia Biotech., isoproterenol, alprenolol, clenbuterol, salmaterol, and ICI-118551 were purchased from MCE. BI-167107 was synthesized by professor Xin Chen at Changzhou University. V2Rpp were synthesized by Tufts University core facility. All of the other reagents were from Sigma.

Primary astrocytes as well as cells of the three clones were collected, washed with PBS, and homogenized in homogenization buffer (0.32 M sucrose; 50 mM sodium phosphate buffer, pH 6.5; 50 mM KCl, 0.5 mM spermine; 0.15 mM spermidine; 2 mM EDTA, and 0.15 mM EGTA), containing protease inhibitors (antipain, 2 μg/mL; aprotinin, 2 μg/mL; benzamidine, 1.0 mM; leupeptin, 2 μg/mL; pepstatin A, 2 μg/mL; phenylmethylsulfonyl fluoride, 1.0 mM, Sigma-Aldrich, St. Louis, MO, USA). Total protein concentration was determined by the Ouant-iT™ protein assay using a Qubit™ fluorometer (Invitrogen, Carlsbad, CA, USA). Equal amounts of proteins (10–20 μg) were loaded onto each lane of 12% polyacrylamide-SDS denaturing gels. After electrophoresis, samples were blotted onto PVDF membranes (0.45 μm pore-size, Amersham Biosciences, Little Chalfont, United Kingdom). Correct transfer of proteins to the membrane, and concentrations of the samples were visualized by staining with Ponceau red for 5 min. Finally, membranes were immunostained with a mouse monoclonal anti-H1.0 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary anti-mouse antibody was from Sigma-Aldrich (St. Louis, MO, USA).