Mouse monoclonal anti γ tubulin

Purified PBMs were infected with BeninΔA179L-mNG and Benin-mNG at an MOI of 0.1. Cells were collected into tubes at 4, 8, 16, and 24 hpi and centrifuged at 400 × g for 10 min at 4°C. Cells were lysed in 4× Laemmli Sample Buffer (Bio-Rad) supplemented with 10% β-mercaptoethanol. Cell lysates were heated at 100°C for 5 min before running on a 12% mini-Protean TGX gel (Bio-Rad). After electrophoresis, protein was transferred to Amersham Hybond P 0.45 polyvinylidene difluoride (PVDF) blotting membranes (Cytiva). Blots were blocked with 5% blotto (5% milk in 0.1% Tween in 100% Tris-buffered saline (TBS)) and probed for ASFV p30 mouse monoclonal antibody and a cellular loading control, mouse monoclonal antiγ-tubulin (Sigma-Aldrich). After probing with either a secondary HRP-conjugated goat antimouse IgG antibody (CST) or the IRDye 800CW goat antimouse secondary antibody (LI-COR), target antibodies were detected via ECL Select (Amersham) or imaged on the Odyssey CLx Infrared imaging system (LI-COR), respectively.

Eye antenna discs and brains from third-instar larvae, and pupal wings at appropriate developmental stages, were dissected and lysed in protein loading buffer. Lysates from eight discs or wings, or two brains, were loaded per lane for SDS-PAGE analysis and western blots were performed using standard procedures. Antibodies: Guinea pig polyclonal anti-Pk[C] (1:1,000 dilution; the same antibody used for immunostaining), mouse monoclonal anti-γ-Tubulin (1:1,000 dilution, Sigma-Aldrich, T6557), rat monoclonal anti-HA (clone 3F10, 1:1,000 dilution; the same antibody used for immunostaining), mouse monoclonal anti-Arm (1:1,000, DSHB). Secondary antibodies were Peroxidase-conjugated goat anti-guinea pig (1:10,000), goat anti-mouse (1:10,000), goat anti-rat (1:10,000) antibodies (all from Jackson Immuno Research), and detection used SuperSignal West Pico Chemiluminescent Substrate (Thermo-Fisher, 34080)

The following antibodies were purchased: rabbit polyclonal anti STING #19851-a-AP (Proteintech, Rosemont, IL, USA), mouse polyclonal anti-STAT1 A302-753A (Bethyl Laboratories Inc., Montgomery, TX, USA), mouse monoclonal anti p-STAT1 (Y701) sc-136229, mouse monoclonal anti TAP1 (D-11) sc-518133, mouse monoclonal anti TAP2 (B-2) sc-515576, mouse monoclonal anti TPN (TO-3) (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit monoclonal anti beta-2 microglobulin (4H5L6) (Thermo Fisher Scientific) #701250, mouse monoclonal anti γ-tubulin #T6657 (Sigma-Aldrich, St. Louis, MI, USA), FITC mouse monoclonal MHC Class I (H-2Kb) (AF6-88.5.5.3) #11-5958-83, PE mouse monoclonal MHC Class I (H-2Kb) (AF6-88.5.5.3) # 12-5958-82, PE mouse monoclonal SIINFEKL/H-2Kb (eBio25-D1.16) #12-5743-82, PE mouse monoclonal IgG1 kappa isotype control (P3.6.2.8.1) #12-4714-42, FITC mouse monoclonal IgG2a kappa (eBM2a) #11-4724-81 (eBioscience^TM, Thermo Fisher Scientific), and horseradish peroxidase (HRP)-conjugated secondary antibodies GtxRb-003-DHRPX and GtxMu-003-DHRPX (Immuno Reagents Inc., Raleigh, NC, USA).