Secondary peroxidase labeled anti mouse or anti rabbit immunoglobulin g antibodies

Cells were harvested, fractionated and transferred onto nitrocellulose membranes as described previously (Kim et al., 2015 (link)). Antibodies to STING (D2P2F), phospho-TBK1 (D52C2) and LC3B (D11) were purchased from Cell Signaling Technology. An anti-FLAG M2 antibody (#200474) was purchased from Agilent technologies. Antibodies to tubulin (B-5-1-2) and TBK1 (AOW9) were purchased from Sigma–Aldrich and EMD Millipore, respectively. Anti-HCMV IE (CH160) antibody was purchased from Virusys. Antibodies to IE86 (12E2), c-Myc (9E10) and GST (B-14) were purchased from Santa Cruz Biotechnology. Secondary peroxidase-labeled anti-mouse or anti-rabbit immunoglobulin G antibodies were purchased from Jackson ImmunoResearch. The signal intensity of protein bands were quantitated using Image Lab^TM software (Bio-Rad Laboratories).

Cells were harvested, and culture supernatants were concentrated 30-fold using Amicon filter. Equal amounts of proteins were electrophoresed on Mini-PROTEAN^® TGX™ gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to nitrocellulose membranes as described [45 (link)]. Antibodies to NLRP3 (D2P5E), caspase-1 (D7F10) and IL-1β (3A6) were purchased from Cell Signaling Technology; antibodies to ASC (F-9) and c-Myc (9E10) were purchased from Santa Cruz Biotechnology; an anti-Tubulin (T5168) antibody was purchased from Sigma-Aldrich. Secondary peroxidase-labeled anti-mouse or anti-rabbit immunoglobulin G antibodies were purchased from Jackson ImmunoResearch Laboratories, West Grove, PA, USA.