Anti mouse igg hrp

Cell lysates were prepared in NP-40 (Fisher Scientific) or RIPA buffer (MilliporeSigma, St. Louis, MO, USA) containing protease inhibitors (Fisher Scientific). Primary antibodies used were: c-Myc (5606S, Cell Signaling, 1:1,000), GAPDH (2118S, Cell Signaling, 1:1,000), vinculin (v4505, MilliporeSigma, 1:10,000), Chk1 (A300-298AT, Bethyl, 1:5,000), BRD2 (5848, Cell Signaling, 1:1,000), BRD4 (13440, Cell Signaling, 1:1,000), cleaved PARP (5625, Cell Signaling, 1:1,000) and γH2AX (9718S, Cell Signaling, 1:1,000). Secondary antibodies used were: HRP goat anti-rabbit IgG (6721, Abcam, 1:50,000) and HRP anti-mouse IgG (7076, Cell Signaling, 1:5,000). Immunoblots were quantitated using ImageStudio Lite 5.2. Data were first normalized to respective loading controls and then to DMSO control.

Cell lysates (15 μg of protein/well) were loaded onto a SDS-polyacrylamide gel and transferred onto a polyvinylidene difluride membrane. Arg-1 and iNOS were detected by blocking the membrane for 1 h in 5% nonfat dried milk or 5% BSA at room temperature followed by overnight incubation in primary Ab at 4°C and 1 h incubation with HRP anti-mouse IgG (Cell Signaling, Danvers, MA, USA) or HRP-anti rabbit IgG (GE Healthcare, UK) at room temperature. ECL Plus (Perkin Elmer, Waltham, MA, USA) was used for detection. The following primary antibodies were used: Arg-1 (BD Pharmingen), iNOS (Cell Signaling), and GAPDH (Millipore, Billerica, MA, USA).

ELISAs were performed as previously described(20 , 47 ) and reproduced here for convenience. Briefly, Triton X-100 and RNase A were added to serum samples at final concentrations of 0.5% and 0.5mg/ml respectively and incubated at room temperature (RT) for 30 minutes before use to reduce risk from any potential virus in serum. 96-well MaxiSorp plates (Thermo Scientific #442404) were coated with 50 μl/well of recombinant SARS CoV-2 S1 protein (ACROBiosystems S1N-C52H3) and RBD (ACROBiosystems SPD-C52H3) at a concentration of 2 μg/ml in PBS and were incubated overnight at 4 °C. The coating buffer was removed, and plates were incubated for 1h at RT with 250 μl of blocking solution (PBS with 0.1% Tween-20, 3% milk powder). Serum was diluted in dilution solution (PBS with 0.1% Tween-20, 1% milk powder) and 100 μl of diluted serum was added for two hours at RT. Plates were washed three times with PBS-T (PBS with 0.1% Tween-20) and 50 μl of HRP anti-mouse IgG (Cell Signaling Technology #7076, 1:3,000) diluted in dilution solution added to each well. After 1 h of incubation at RT, plates were washed three times with PBS-T. Plates were developed with 100 μl of TMB Substrate Reagent Set (BD Biosciences #555214) and the reaction was stopped after 15 min by the addition of 2 N sulfuric acid. Plates were then read at a wavelength of 450 nm and 570nm.