Anti glyceraldehyde 3 phosphate dehydrogenase gadph

Bone marrow-derived dendritic cells (BMDCs) were generated according to the protocol (Dai et al., 2014). BMDCs (1 × 10⁶) from WT and KO mice were infected with MVA at a MOI (multiplicity of infection) of 10 or an equivalent amount of Heat-MVA, or UV-MVA. Whole-cell lysates were prepared. Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the polypeptides were transferred to a nitrocellulose membrane. Phosphorylation of IRF3, IRF3, and STING levels were determined using respective antibodies (Cell Signaling). Vaccinia E3 protein level was determined by using anti-E3 monoclonal antibody (MAb 3015B2) kindly provided by Dr. Stuart N. Isaacs (University of Pennsylvania) (43 (link)). Anti-glyceraldehyde-3-phosphate dehydrogenase (GADPH) or anti-β-actin antibodies (Cell Signaling) were used as loading controls. B16-F10 melanoma cells were infected with MVA at a MOI of 10 or with an equivalent amount of Heat-MVA. Cell lysates were collected at various times post infection. Western blot analysis was performed using anti-phospho-IRF3, anti-IRF3, and anti-GAPDH antibodies as described above.