Hrp conjugated anti rabbit antibody

Leaf discs were incubated overnight in water then treated with 100nM elf18 and samples were frozen in liquid nitrogen. Samples to test for a potential constitutive activation of MAPKs were frozen directly from the plant. Proteins were extracted by grinding samples and boiling for 5 min in Laemmli sample buffer. Protein extracts were run on 10% SDS PAGE gel and transferred onto PVDF membrane. Western blots were performed with anti-p44/42 MAPK (Cell Signaling Technology) and HRP conjugated anti-rabbit antibodies (Sigma).

Mouse monoclonal anti-RABV-G E559 and rabbit polyclonal anti-P serum P160 were described previously [34, 36 (link)]. Anti-RABV-G 1C5 antibodies were purchased from Abcam. GAPDH was obtained from Santa Cruz Biotechnology. PitStop2, MβCD and EIPA were purchased from Sigma-Aldrich. Alexa Fluor 488 and 594 anti-mouse antibodies were obtained from Molecular Probes. HRP-conjugated anti-mouse and HRP-conjugated anti-rabbit antibodies were purchased from Sigma-Aldrich.

Cells were washed with ice-cold PBS, pelleted, and lysed in 50 mM Tris–HCl at pH 8.0, 150 mM NaCl, 0.5% SDS, and 1% NP-40 supplemented with a Halt Protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and Phosphatase inhibitor cocktails 2 and 3 (Sigma, St. Louis, MO, USA) on ice for 30 min. Lysates were then cleared by centrifugation for 10 min at 14,000× g. Total protein concentration was measured using a DC protein assay (Bio-Rad, Hercules, CA, USA) and 10–50 µg of protein was mixed with loading buffer. For the analysis of H2A and γH2AX, protein samples were separated on 4–15% Mini-PROTEAN^® TGX™ Precast Protein Gels (Bio-Rad) and transferred to a PVDF membrane using Trans-Blot Turbo RTA Mini 0.2 µm PVDF Transfer Kit (Bio-Rad). The primary antibodies used were: Histone H2A Antibody II (#2578, Cell Signaling), Phospho-Histone H2A.X (Ser139) (20E3), and Rabbit mAb (#9718, Cell Signaling). HRP-conjugated anti-rabbit antibody (Sigma) was used as secondary antibody. Immuno-reactive bands were detected on the ChemiDoc MP system (Bio-Rad) using the Clarity Western ECL substrate (Bio-Rad).

To check the correct expression of the HIV-1_BX08 gp120 protein, monolayers of DF-1 cells were mock infected or infected at 5 PFU/cell with the different viruses. At 24 hpi, cell extracts were lysed in Laemmli buffer and fractionated in 8% SDS-PAGE, and then analyzed by Western blotting with rabbit polyclonal anti-gp120 antibody against clade B IIIB strain (CNB; diluted 1:3000) to analyze the expression of the gp120 protein. As loading controls, we used rabbit anti-β-actin (Cell Signaling, Danvers, MA, USA; diluted 1:1000), and rabbit anti-VACV E3 (CNB; diluted 1:1000) antibodies. An HRP-conjugated anti-rabbit antibody (Sigma-Aldrich, St. Louis, MO, USA; diluted 1:5000) was used as the secondary antibody. The immunocomplexes were detected with an HRP-luminol enhanced-chemiluminescence system (ECL Plus) (GE Healthcare, Chicago, IL, USA).

Stimulated PBMCs (5x10⁶) were lysed in 100 µL lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1% Triton X-100, 40 mM α-glycerophosphate, 50 mM sodium fluoride, 200 μM sodium vanadate, 10 μg/mL leupeptin, 423 10 μg/mL aprotinin, 1 μM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride) and stored at -80°C until further use. Lysates were centrifuged (10 min, 14000 rpm, 4°C) to eliminate cell debris. Supernatans were used for Western blot analysis. Bio-Rad 4-15% polyacrylamide gels were used to load equal amounts of protein. Next, the separated proteins were transferred to Nitrocellulose (Bio-Rad) membranes and the membrane was blocked for 1 hour with 5% BSA (bovine serum albumin, Sigma) in TBS-Tween buffer (TBS-T). This was incubated overnight with a primary antibody (RSK1 (D6D5) and Phospho-p90RSK (Thr359) (D1E9), both Cell Signaling or rabbit anti-actin, Sigma) at a dilution of 1:1000 in blocking buffer (TBS-T with 5% BSA). Blots were washed following overnight incubation in TBS-T three times and incubated with HRP-conjugated anti-rabbit antibody (1:5000; Sigma) in 5% milk in TBS-T for 1 hour. Blots were developed with ECL (Bio-Rad) according to the manufacturer’s instructions and results were quantified with Image Lab (version 5.2.1).

Freshly isolated CD4 SP thymocytes from CD11c-cre p28^flox/flox mice and littermates were lysed in lysis buffer (20 mM Tris pH 8.0, 137 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM aprotinin, 1 mM leupeptin, 1 mM EGTA, 1 mM Na3VO4, 1 mM tetrasodium pyrophosphate, and 10 mM NaF). The lysate was resolved on a 12% reducing SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. After blocking with Tris-buffered saline (pH 7.4) containing 5% dried skimmed milk, the membrane was incubated with anti-STAT1. anti-phospho-STAT1, anti-STAT3. anti-phospho-STAT3, or anti-β-actin (R&D Systems, Minneapolis, MN).), followed by probing with HRP-conjugated anti-rabbit antibody (Sigma Aldrich). The immunoreactive bands were detected by chemiluminescence with ECL detection reagents (Life Technologies, Grand Island, NY, USA).