Goat anti mouse igg hrp secondary antibody

Micro-vinyl plates (96-well, Corning Costar, Corning, NY, USA) were coated with 0.5 µg/mL of BaL gp120 Env recombinant protein (NIH AIDS reagent #4961, Germantown, MD, USA) or Gag IIIB recombinant protein (NIH AIDS reagent #3276, Germantown, MD, USA) in sodium bicarbonate buffer and incubated overnight at 4 °C. Plates were washed three times with 0.05% PBST and blocked with 5% BSA in PBS. Mouse serum was diluted at 1:500 in PBS and incubated overnight, then washed 5 times with 0.05% PBST before goat anti-mouse IgG HRP secondary antibody (Cell signaling #7076S, Danvers, MA, USA), diluted 1:20,000 in 5% BSA in PBS, was added to each well for 2 h. After washing 6 times with 0.05% PBST, the 1-Step™ Turbo TMB-ELISA (Thermo Scientific #34022, Rockford, IL, USA) reagent was added and incubated for 20 min at room temperature. Sulfuric acid (2N) was used to stop the reaction. Plates were analyzed on a spectrophotometer and optical density was measured at 450 nm.

The HCT116 cells were seeded in 60 mm dishes overnight and treated with the indicated compounds. Harvested cells were disrupted with cell lysis buffer (50 mM Tris–HCl, pH 8.0; 150 mM NaCl; 0.5% Na-deoxycholate; 1% NP-40; 0.1% SDS), and with protease inhibitor mixture. After sonication, the cell lysates were centrifuged at 14 000g and 4 °C for 15 min. Protein concentration of supernatant was determined. Equal amounts of lysate protein were separated by SDS–polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat skim milk in TBST [20 mM Tris–HCl (pH 7.6), 135 mM NaCl and 0.1% Tween 20], and then incubated with specific primary antibodies to ATM, ATR, and P53 (Cell Signalling Technology, Danvers, MA) at 4 °C overnight. β-Actin was used as a loading control. The next day, the membrane was incubated with goat anti-mouse IgG–HRP secondary antibody (Cell Signalling Technology, Danvers, MA) at room temperature for 2 h. The signals were detected by chemiluminescence utilizing the enhanced chemiluminescent reagent and recorded on the gel imaging system (Tanon, Shanghai, China) and relative quantified using Image J software program. The experiment was repeated three times, and A549 cells were tested in the same way. The test results of A549 cell line are provided in the ESI (Fig. S72†).

Micro-vinyl plates (96-well, Corning Costar, Corning, NY, USA) were coated with 0.5 µg/mL of BaL gp120 Env recombinant protein (NIH AIDS reagent #4961, Germantown, MD, USA) or Gag IIIB recombinant protein (NIH AIDS reagent #3276, Germantown, MD, USA) in sodium bicarbonate buffer and incubated overnight at 4 °C. Plates were washed two times with 0.05% PBST and blocked with 5% BSA in PBS. Duplicate wells containing mouse serum diluted at 1:500 in PBS were incubated overnight. The following day, diluted serum was removed and one of the duplicate wells was treated with 7M urea for 20 min, while the other well was treated with 0.05% PBST. After incubation, wells were washed 4 more times with 0.05% PBST, then incubated with goat anti-mouse IgG HRP secondary antibody (Cell signaling #7076S, Danvers, MA, USA), diluted 1:20,000 in 5% BSA in PBS for 2 h. After washing 6 times with 0.05% PBST, the 1-Step™ Turbo TMB-ELISA (Thermo Scientific #34022, Rockford, IL, USA) reagent was added and incubated for 20 min at room temperature. Sulfuric acid (2N) was used to stop the reaction. Plates were analyzed on a spectrophotometer and optical density was measured at 450 nm. The avidity index was calculated by taking the OD450 value of the urea treated well divided by the untreated well, as previously described [40 (link),41 (link)].