Anti α tubulin

Cells were lysed in lysis buffer composed of 25 mmol/L Tris-HCl, pH8.0, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5% IgepalCA-630, and protease and phosphatase inhibitors (Roche). The cell lysates were centrifuged (15,000 × g), at 4°C for 5 min. For co-immunoprecipitation assay, cell lysates were immunoprecipitated for 1 to 2 h with FLAG M2 Affinity Gel for FLAG-tagged proteins or anti-HA antibody followed by protein G/A-Agarose Suspension (EMD Millipore) for HA-tagged proteins. Immunocomplexes were then washed three times. All samples were denatured in 1x sample buffer (50 mmol/L Tris-HCl, pH6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue) for 5 min at 100°C. Proteins were electroblotted onto nitrocellulose membranes (GE Healthcare). The following primary antibodies were used: anti-LMP2 and anti-LMP7 from Santa Cruz Biotechnology, anti-α-tubulin from Developmental Studies Hybridoma Bank, anti-STAT1, anti-p-STAT1, anti-MAVS, anti-STING, anti-IRF3, and anti-p-IRF3 from Cell Signaling Technology, anti-p84 from GeneTex, anti-FLAG M2 from Sigma, anti-HA and anti-Myc from BioLegend. HRP-conjugated secondary antibodies (Thermo Fisher Scientific) were used for detection with Western ECL substrates (Thermo). Chemiluminescent signals were detected by film or ChemiDoc (Bio-Rad). Quantification was performed with ImageJ software.^{48 (link)}