Peroxidase conjugated affinipure goat anti mouse igg

Cultures were grown in bactopeptone medium at 30°C and 250 rpm, and culture supernatants were collected by centrifugation at t2. SDS-PAGE was carried out using 12% acrylamide gels, and silver stained according to Blum et al.[42] . Proteins were blotted onto Immun-Blot PVDF membranes (Bio-Rad), and nonspecific binding was blocked with 5% nonfat milk. The HblB (binding) component was detected using monoclonal antibody 2A3 diluted 1∶15 [43] (link), kindly provided by Dr Erwin Märtlbauer (Ludwig-Maximilians-Universität, Munich, Germany). Peroxidase-conjugated AffiniPure Goat-anti-mouse IgG (Jackson Immuno Research Laboratories Inc) were used at 0.8 µg/ml as secondary antibody, and bands were detected using the SuperSignal West Femto Substrate (Pierce) and quantified using ImageJ [41] . After subtraction of the background, the mean gray value for each band was normalized to the intensity of the band in the 407 wild type sample, arbitrarily defined as 1.

Cell lysates were obtained in cell lysis buffer [50 mmol/L Tris, HCl (pH 7.4), 150 mmol/L NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 1% (w/v) sodium deoxycholate and protease/phosphatase inhibitors (Complete, Roche Diagnostics, Risch-Rotkreuz, Switzerland)], and total protein concentration in each sample was measured using a Bicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies were as follows: p-eIF2α (Abcam, Cambridge, England, ab32157), eIF2α (Cell Signaling technology, Danvers, MA, USA, #9722), CHOP (Santacruz, Dallas, TX, USA, sc-575), ATF4 (Cell Signaling technology, Danvers, MA, USA, #11815), HSP70 (Enzo Life Sciences, Farmingdale, NY, USA, SPA822), cleaved Caspase-3 (Cell Signaling technology, Danvers, MA, USA, #9661), PARP (Cell signaling technology, Danvers, MA, USA, #9542), and α-tubulin (Sigma, St. Louis, MO, USA, T9026). Secondary antibodies were as follows: Peroxidase-conjugated affinipure goat anti-mouse IgG (Jackson laboratory, Bar harbor, ME, USA, 115-035-003) and anti-rabbit IgG (Jackson laboratory, Bar harbor, ME, USA, 111-035-003). Chemiluminescence detection using enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) was performed. Membranes were exposed to imaging film and developed using an automatic film processor (Agfa, Mortsel, Belgium, CP1000).