Anti mouse horseradish peroxidase hrp conjugated secondary antibody

Frozen pellets were lysed in RIPA buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM TCEP, and protease and phosphatase inhibitors (Roche)). Cell lysates were sonicated and then centrifuged at 4°C at 16 000 g for 10 min. LDS sample buffer (Life Technologies) and tris(2-carboxyethyl)phosphine (TCEP), was added to samples at a final concentration of 1× and 25 mM, respectively, and samples were boiled for 10 min. Samples were loaded on a SDS–PAGE gel (NuPAGE precast gels, Life Technologies) and were then transferred to nitrocellulose membranes (Whatman). Blots were probed with the following antibodies: SMC1 (Bethyl Laboratories), phospho-ERK1/2 (p-p44/42 MAPK T202/Y204, Cell Signalling and Technology) and pan-ERK1/2 (p44/42 MAPK, Cell Signalling and Technology), followed by anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Scientific). Chemiluminescence was measured using an Odyssey Fc Imaging System (Licor).

Cells were harvested in RIPA buffer (Sigma‐Aldrich) supplemented with a cocktail of protease inhibitors. Proteins were resolved on SDS‐polyacrylamide gels and electro‐blotted onto PVDF membranes (Millipore) or nitrocellulose membranes (Biorad). Membranes were incubated with following rabbit anti‐human primary antibodies: cyclin D1 (#2922S; Cell Signaling, 1:1000, lot:3), actin (Sigma‐Aldrich, 1:1000), HSP90 (#4877; Cell Signaling, 1:2000), FOXO3A (#2497; Cell Signaling, 1:1000), PHD1 (NB100‐310; Novus Biologicals, 1:500), phospho‐histone H2AX (Ser139; #9718; Cell Signaling, 1:1000) and mouse anti‐human primary antibody CtBP (sc‐17759; Santa Cruz, 1:1000) at 4°C overnight, washed in PBS with 0.05% Tween 20, and incubated for 1 hour with goat anti‐rabbit or goat antimouse horseradish peroxidase (HRP)–conjugated secondary antibody (ThermoFisher Scientific). HRP activity was detected with an ECL detection kit (Pierce, ThermoFisher Scientific).

Neat sputum samples were denatured in the presence of 2.5% β-mercaptoethanol at 95 °C for ~ 10 min and were subjected to Western blotting. In brief, samples were transferred to PDVF membranes and blocked using 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST-T). For detection of neutrophil elastase, membranes were probed using a mouse-monoclonal anti-hELA2 antibody, raised against residues M1 - N252 (1:3000, R&D systems), primary antibodies were detected using an anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (Thermo-Fisher Scientific). Membranes were then stripped, re-blocked and re-probed for SPLUNC1 using a goat polyclonal hPLUNC1 antibody raised against residues Q20 - V256 of hPLUNC1 (1:3000, R&D systems), a secondary anti-goat HRP (Thermo-Fisher Scientific) conjugated antibody was used for detection of hPLUNC1. Secondary antibodies were detected by enhanced chemiluminescence (Thermo-Fisher Scientific).