Swine α rabbit hrp

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS, Bodinco DV), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. BHK-21 cells were cultured in Glasgow’s minimal essential medium (GMEM, Gibco) supplemented with 8% (v/v) FCS, 10% (v/v) tryptose phosphate broth (TPB), 10 mM HEPES pH 7.4, 100 U/ml penicillin, and 100 mg/ml streptomycin. MARC-145 were cultured in DMEM supplemented with 8% (v/v) FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. DMEM and cell culture supplements were obtained from Lonza.
Primary antibodies that were used were mouse-α-β-actin (A5316, Sigma-Aldrich), rabbit-α-GFP [9 (link)], mouse-α-V5 (37–7500, Invitrogen), mouse-α-myc (9E10, Roche), mouse-α-HA (ab18181, Abcam), and mouse-α-FLAG (F1804, Sigma-Aldrich). As secondary antibodies, goat-α-mouse-HRP (P0447, Dako), swine-α-rabbit-HRP (P0217, Dako), goat-α-mouse-AlexaFluor488 (A11001, Life Technologies), and donkey-α-rabbit-Cy3 (711-165-152, Jackson). The antibody α-ORF7 (P3-05-A27, MSD Animal Health) was obtained using mouse antiserum. The antibody α-nsp2/3 LV was obtained using rabbit antiserum [13 (link)].

For immunoblot analysis BOECs were subjected to flow as described. Next, BOECs were washed 3× with PBS, lysed in 2x sample buffer (125 mm Tris, 2% SDS, 20% glycerol, 0.02% Bromphenol blue, 20 mm DTT), boiled for 10 min, passed 5× through a 29G needle, spun down 15 min at 16,000 × g, and filled up to 1× loading buffer. An estimated 20 μg sample was loaded per well on a NuPAGE 3–8% Tris-Acetate gel (ThermoFisher Scientific). Proteins were separated and transferred to an iBlot nitrocellulose blot (ThermoFisher Scientific). Blot was blocked for 1 h in blocking buffer (4% BSA, 0.1% Tween-20 in TBS (Tris-buffered saline), incubated overnight in Sheep polyclonal αLAMA4 (AF7340, R&D systems) in blocking buffer, washed 3× with TBS 0.1% Tween-20, incubated 1 h in Donkey α Sheep-HRP (713–035-147, Jackson ImmunoResearch) in blocking buffer, and developed using BM chemiluminescence blotting substrate (Roche). The loading control was stained using Rabbit IgG α α-tubulin (Ab52866, Abcam) and Swine α Rabbit-HRP (P0399, DAKO).