Horseradish peroxidase conjugated anti rabbit igg antibody

Western blotting was used to determine intracellular OPTN levels. Cells were lysed in Laemmli sample buffer (0·06 m Tris–HCl pH 6·8, 2% SDS, 10% glycerol, 5% β₂-mercaptoethanol (VWR, Leicestershire UK), 0·04% [weight/volume (w/v)] bromophenol blue, Complete protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany), and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich). Proteins were separated by SDS–PAGE and transferred onto Hybond P nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) with a semi-dry transfer system (Trans-Blot SD semi-dry transfer cell; Bio-Rad, Hemel Hempstead, UK) in transfer buffer [200 mm glycine, 0·1% SDS (w/v), 10% methanol (v/v), 25 mm Tris–HCl pH 8·8]. Membranes were incubated overnight at 4° with primary antibodies directed against OPTN (HPA003360; Sigma-Aldrich, 1 : 1000) or β-actin (Sigma-Aldrich; 1 : 1000) and then with horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare, Amersham, UK, 1 : 2000) for 1 hr at room temperature, and developed using the Enhanced Chemiluminescence method (ECL PLUS; GE Healthcare). Densitometry analysis was performed using imagej software (National Institutes of Health, Bethesda, MA), with band densities normalized to β-actin.

ApoB levels in plasma were measured by ELISA. Chicken monoclonal anti-apoB antibody (HUC20, Hiroshima Bio-Medical Co., Ltd., Hiroshima, Japan) was used for capture and rabbit anti-apoB antiserum (Abcam plc., Cambridge, UK) was used for detection. Capture antibody was coated on a Costar 96 well EIA/RIA Easy Wash Clear Flat Bottom High Binding Plate (Corning Incorporated, Corning, NY) overnight at 4°C, and a blocking buffer (1× Reagent Diluent Concentrate 2, R&D Systems, Inc., Minneapolis, MN) was plated to inhibit nonspecific binding for 2 hr at room temperature. All the following steps were performed at room temperature. Diluted samples were then added to the plate and incubated for 2 hr. Next, the detection antibody was incubated for 2 hr, followed by an incubation of horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare) for 2 hr. Wells were washed with PBS plus 0.05% Tween 20 for three times after all the incubation steps. TMB (3, 3′, 5, 5′-tetramethylbenzidine, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) substrate was added to the plate, and the reaction was stopped with 2 N sulfuric acid (Nacalai tesque). Absorbance at 450 nm was measured on a plate reader ARVO X3, and absorbance at 544 nm was subtracted to normalize background. A standard curve was prepared with plasma from mice at E18.5–E19.5 for the relative quantification.

Anti-XoxF and anti-MxaF antisera were generated against synthesized peptides corresponding to residues 339–351 of MxaF and residues 374–387 of XoxF1, respectively. Whole-cell lysates of M. trichosporium OB3b and OB3b ΔmxaF mutant were separated on 10% (w/v) acrylamide gel and transferred to a PVDF membrane following general protocols. The Precision Plus Protein™ Dual Color Standards (Bio-Rad Laboratories, Hercules, CA, United States) were used for the molecular weight marker. The blotted membrane was blocked with 5% (w/v) skim milk at room temperature for 1 h and treated at room temperature for 1 h with anti-MxaF or anti-XoxF1 antisera at 1:2,000 dilution in Tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20 (TBS-T; Calbiochem, San Diego, CA, United States). Membrane-bound MxaF and XoxF1 were detected with horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare, Little Chalfont, United Kingdom) at 1:10,000 dilution in TBS-T and visualized using EzWestLumi Plus (ATTO Corp., Tokyo, Japan) according to the manufacturer’s instructions.

The degree of STAT3 activation was determined by measuring the increased expression of phosphorylated STAT3 (pSTAT3) with Western blot analysis, as previously described15 (link). Briefly, the solubilized cells were run on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) transfer membrane (Millipore, Bedford, MA, USA). To detect pSTAT3, the membranes were exposed to an anti-pSTAT3 antibody (D3A7; Cell Signaling Technology Japan, Tokyo, Japan) and visualized using a horseradish peroxidase-conjugated anti-rabbit IgG antibody with ECL Western blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA). To detect STAT3, the membranes were exposed to anti-STAT3 antibody (sc-8019; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and visualized using a horseradish peroxidase-conjugated anti-mouse IgG antibody with ECL Western blotting detection reagent. The membranes were re-blotted with an anti-β-actin antibody (C4) (sc-47778; Santa Cruz Biotechnology, Inc.) as an internal calibration control.