Horseradish peroxidase hrp conjugated antibody

Cells were washed and lysed directly in the cell culture well in 2× Laemmli sample buffer (Bio-Rad). The cell lysates were then sonicated and heated for 10 min at 70°C, and aliquots were stored at −20°C. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham). Membranes were incubated with the primary ATM (2873), p-ATM (5883), p-ATR, Chk1 (2360), and p-Chk1 (2348) antibodies from Cell Signaling; p-RPA32 (A300-246) antibody from Bethyl; and lamin A/C (SAB4200236) and ATR (SAB4200348) antibodies from Sigma. Secondary anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies (Jackson Laboratories) were visualized with chemiluminescence Clarity Western ECL substrate (Bio-Rad) and imaged using a ChemiDoc XRS imager and Image Lab software (Bio-Rad).

DADS (Purity of 99%) was purchased from Tengzhou wutong aroma chemicals Co., Ltd. (Tengzhou, Shandong, China). Total superoxide dismutase (T-SOD), Malondialdehyde (MDA), glutathione peroxidase (GSH-PX), glutathione (GSH), myeloperoxidase (MPO) and total antioxidant capacity (T-AOC) commercial reagent kits were purchased from Nanjing Jiancheng Biology Engineering Institute (Nanjing, Jiangsu, China). Enzyme-linked immuno sorbent assay (ELISA) commercial kits for TNF-α, IL 1β and IL-6 were bought from Shanghai MultiSciences (Lianke) Biotech Co., Ltd. (Shanghai, China). DAB Detection Kit was purchased from Zhongshan Goldebridge Biotechnology CO., Ltd. (Beijing, China). Anti-NF-κB p65, i-κB, and NQO1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Nrf2 antibodies were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH antibodies were provided by Proteintech Biotechnology (Rocky Hill, CT, USA). Horseradish peroxidase (HRP)-conjugated antibodies was bought from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, USA). Rabbit polyclonal antibodies against rat CD4⁺, CD8⁺, MMP-9 and TIMP-1 were purchased from Abcam (Shanghai, China). Other reagents used in the experiment were all of analytical grade.

The primary antibodies (Ab) used were the mouse monoclonal (MAb) anti-DBP, B6 [52 (link)]; the rabbit polyclonal anti-DBP (a kind gift of T. Dobner); the mouse MAb anti-E1B 55kDa, 2A6 [53 (link)]; the mouse MAb anti-p53 (DO-1, Santa Cruz Biotechnology); the mouse polyclonal anti-Mre11 (Novus Biologicals); the mouse MAb anti-β actin (Santa Cruz Biotechnology); and the mouse MAb anti-fiber (Abcam). The secondary antibodies used were anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies (both from Jackson ImmunoResearch), anti-mouse Alexa Fluor 568, and anti-rabbit Alexa Fluor 488 (both from Invitrogen).

Antibodies directed against PRAS40 (# 701075) and phospho-PRAS40 (Thr 246) (# 701058) were purchased from Life Technologies (Carlsbad, California). Antibodies against phospho-p53 (Ser 15) (# 9286), phospho-p53 (Ser 46) (# 2521) and BAX (# 2772) were from Cell Signaling (Ozyme, Saint Quentin en Yvelines, France). Antibodies directed against p53 (# Sc-126) and β-actin (# Sc-1616) were purchased from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated antibodies were obtained from Jackson ImmunoResearch (Bar Harbor, ME).

Cells were lysed in whole cell lysis buffer (100 mM Tris‐HCl pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, 0.1% (w/v) bromophenol blue, 2.5% (v/v) β‐mercaptoethanol). Proteins were separated by SDS‐PAGE and transferred to nitrocellulose membranes (GE Healthcare, Munich, Germany), which were then blocked in 5% (w/v) non‐fat milk dissolved in PBS containing 0.1% (v/v) Tween 20 (Sigma‐Aldrich, P5927). Membranes were probed with specific antibodies against: caspase‐9 (Cell Signaling Technology (CST), Danvers, MA, USA, #9508), FADD (Santa Cruz, Dallas, USA, sc‐6036), cleaved caspase‐8 (CST, #8592), caspase‐8 (CST, #4790), caspase‐3 (CST, #9662), Atg5 (CST, #12994), light‐chain 3 (LC3) (Sigma‐Aldrich, L7543), p‐eIF2α (CST, #3398), eIF2α (CST, #5324), ATF4 (CST, #9508), CHOP (CST, #2895), FLAG (Sigma, F1084) and actin (Sigma‐Aldrich, A2066). Horseradish peroxidase (HRP)–conjugated antibodies were from Jackson ImmunoResearch, Ely, UK: anti‐rabbit (111–035–003), anti‐mouse (115–035–003) and anti‐goat (705–035–003). The signal was visualized using enhanced chemiluminescence reagent (PerkinElmer, Waltham, Massachusetts, USA, NEL102001EA) or ImmunoCruz Luminol Reagent (Santa Cruz, sc‐2048).

Bacteria collected from different treatments were grown to the log phase, disrupted using a sonicator (BD, Franklin Lakes, NJ, USA), and precipitated with trichloroacetic acid and acetone. Protein concentration was detected with a BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Then, 25 µg protein samples were separated on 10% SDS-polyacrylamide gel and electrophoresed at 100 V for 80 min. Samples were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-rad, Hercules, CA, USA) with 350 mA for 90 min. The membrane was probed with an antibody against PBP2a (RayBiotech, Norcross, GA, USA). Anti-GAPDH (Pierce, Rockford, IL, USA) was used to ascertain equal loading. Horseradish peroxidase (HRP)-conjugated antibody (Jackson immunoresearch, West Grove, PA, USA) and the enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) were used for evaluation.