Mem per plus kit

The membrane proteins from BHK-21 cells were isolated using Mem-PER Plus Kit (Thermo) in accordance with the manufacturer’s protocol. VOPBA was performed as essentially described in Thepparit and Smith (2004) (link). Briefly, membrane proteins were subjected to electrophoresis through 12% SDS-PAGE and transferred to PVDF membranes. The membrane containing transferred proteins was blocked with 5% BSA in PBST at 37°C for 2 h. For viral overlay, the membranes were incubated with 10⁵ TCID₅₀ of TMUV in 5% BSA in PBST overnight at 4°C and washed three times with PBST buffer. Subsequently, the membranes were incubated with monoclonal antibody against TMUV at 37°C for 1 h followed by incubated with a alkaline phosphatase-conjugated goat anti-mouse IgG. Finally, the signal was developed using Alkaline Phosphatase Assay Kit (Beyotime) in accordance with the manufacturer’s protocol.

CelLytic M was used to extract protein from cultured cells and brain tissues. Membrane proteins were extracted using the Mem-PER Plus Kit (89842; Thermo Fisher Scientific). Protein samples prepared for SDS-PAGE analysis were denatured by heating in the presence of loading buffer containing 2% SDS (20 mM BME and 100 mM DTT, except in Figure 2A) and separated on a handmade SDS-PAGE gel. Protein samples used for native-PAGE were prepared in loading buffer containing 0.1% SDS (but not BME and DTT) to reduce the dispersion of the EAAT2 bands, after which they were separated on a precast 12% native-PAGE gel (PG01210-N; Solarbio). The following antibodies were used: mouse anti–β-Actin (66009-1-Ig) and rabbit anti-EAAT2 (22515-1-AP), anti-SP1 (21962-1-AP), anti-ubiquitin (10201-2-AP), and anti-AMFR (16675-1-AP) from Proteintech; mouse anti-EAAT2 (sc-365634) from Santa Cruz Biotechnology; and mouse monoclonal anti-FLAG M2 (F1804) from Sigma-Aldrich.

For western blots, indicated cells were lysed in NP-40 lysis buffer with protease and phosphatase inhibitors and immediately snap-frozen. Lysates were separated on 10% SDS-PAGE gels, transferred to PVDF membranes, and probed with primary antibodies overnight and secondary antibodies for 1 h. Membranes were developed using Pierce ECL WB Substrate (Thermo Scientific) and imaged using ChemiDoc Touch (Bio-rad). Where indicated, band intensity was quantified with ImageLab software. For immunoprecipitation, B cell membrane proteins were isolated using MEM-PER Plus kit (Thermo Scientific), then incubated with blocked SNA-agarose beads (Vector Laboratories) overnight. Beads were extensively washed and immunoprecipitate eluted by boiling in denaturing and reducing conditions, before western blot analysis. Uncropped Western blot images are included in Supplementary Figure 8.

Tissue homogenisation and western blotting analysis were performed using conventional methods^{14 (link),31 (link),32 (link)}. Isolation of membrane and cytoplasmic protein fractions were performed following the manufacturer’s protocols (Fermentas, ProteoJET; Thermo, Mem-PER Plus kit). RNA purification, reverse transcription reaction, and real-time qPCR analysis (Takara Bio, Thermal Cycle Dice (TP800), Thermal Cycle Dice Real Time System Ver. 5. 11B) were performed by conventional methods^{14 (link),31 (link),32 (link)}. Briefly, to detect miRNAs, cDNA was generated using an miScriptReverse Transcription Kit (QIAGEN). Expression of miR-16, miR-23 and miR-21 was detected using an miScript SYBR Green PCR Kit (QIAGEN) with the following primers: 5'- TAGCAGCACGTAAATATTGG-3' for miR-16, 5'-ATCACATTGCCAGGGATTTCC-3' for miR-23 and 5'-TAGCTTATCAGACTGATGTTGA-3' for miR-21.