Horseradish peroxidase linked secondary antibody

Western blots were performed as described^{42 (link)}. For protein preparation, cells or tissues were solubilized in a lysis buffer. Protein concentration was determined using a Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA). Then, 20 μg of the protein was electrophoresed by SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). After blocking with 5% skimmed milk in Tris-buffered saline containing Tween 20 (0.1%) for 1 h, the membrane was incubated with polyclonal antibodies against phospho-ERK (1:1,000; Cell Signaling Tech., Danvers, MA, 4376S), ERK (1:1,000; Cell Signaling Tech., 4695S), Glut1 (1:1,000; AB chem, Cambridge, UK, ab652), adiponectin (1:1000; Cell Signaling Tech., 2789) or β-actin (1:1,000; Cell Signaling Tech., 4967S) at 4 °C with gentle shaking overnight. Antibodies were detected by horseradish peroxidase-linked secondary antibody (1:10,000; Cell Signaling Tech., 7074S), using the enhanced chemiluminescence Western Blotting Detection System (Molecular Imager Gel Doc XR System, BIO-RAD). The intensities of the protein bands were performed with ImageJ 1.52v software. Images of original western blotting are shown in Supplementary Figs. 1–3.

Protein extraction and Western blot were done as described previously [29 (link)]. Samples containing equal amounts of protein (30 μg) were separated by electrophoresis on polyacrylamide SDS gels and transferred to polyvinylidene difluoride (PVDF, Amersham, UK) membranes by electroblotting. Subsequently, the membranes were saturated with 5% (v/v) FBS and probed with primary antibodies overnight at 4°C. Membranes were then extensively washed and probed with horseradish peroxidase-linked secondary antibody (Cell Signaling Technologies, Beverly, MA, USA), followed by enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) detection. Membranes were stripped and reprobed for total protein, GAPDH, or α-tubulin to demonstrate equal loading. The values of band intensities were quantified by Quantity One 4.6.2 software (Bio-Rad Laboratories, Hercules, CA, USA) to the respective protein loading controls. All immunoblots shown here are representatives of at least three independent experiments.

To extract protein from hMSCs, the cells were lyzed using RIPA buffer composed of 50 mM Tris–HCl (pH 7.5), 0.25% Na-deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA). After centrifugation at 14,000 rpm for 10 minutes, the supernatant was collected. Protein concentration was measured using the BCA Protein Assay kit (Pierce, Rockford, IL, USA). A 40-μg protein sample was loaded into each lane of a 10% polyacramide gel (Bio-Rad) for electrophoresis, and the separated proteins were then transferred from the gel onto a polyvinylidene fluoride membrane (Bio-Rad). The membrane was incubated with primary antibodies against AKT, phospho-AKT (Ser473), and glyceraldehyde 3-phosphate dehydrogenase (Cell Signaling, Danvers, MA, USA) in a blocking solution composed of Tris-buffered saline containing 5% nonfat milk (Bio-Rad) and 0.1% Tween 20 (Sigma-Aldrich) overnight at 4°C. After removing unbound antibodies, the membrane was incubated with horseradish peroxidase-linked secondary antibody (Cell Signaling) in the blocking solution for 1 hour at room temperature. The immuno-detected protein bands on the membrane were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce), and then documented by the Kodak Image Station 4000R Pro system (Kodak, Rochester, NY, USA).

Samples were harvested, washed in phosphate-buffered saline and after pelleting, lysed directly in lysis buffer (10 mm HEPES/KOH pH7.4, 10 mm KCl, 0.05% NP-40, 1 mm sodium orthovanadate). After centrifugation of the lysate, the supernatant containing the cytoplasmic components was retained, and the pelleted nuclei were washed with lysis buffer before extraction of the nucleoplasmic protein fraction in low salt lysis buffer (10 mM TRIS/HCl pH7.4, 0.2 mm MgCl₂). After blocking, membranes were probed with antibodies against actin (AC-40; Sigma), R26cit (ab19847; Abcam), R2,8,17cit (ab5103; Abcam), PADI2 (ab16478; Abcam) or PADI4 (ab128086; Abcam), followed by the appropriate horseradish peroxidase-linked secondary antibody (Cell Signalling, NEB, Hitchin, UK) and visualised using ECL Prime (GE Healthcare, Amersham, UK).

Equal amounts of protein extracts were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system, electrophoresed, and transferred to nitrocellulose membranes (Amersham). After blocking with 5% (w/v) nonfat milk in PBS for 2 hours at room temperature, the membranes were incubated with specific antibodies overnight, followed by incubation with horseradish peroxidase-linked secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. The signals were detected by the chemiluminescence phototope-HRP kit (Millipore), according to the manufacturer’s instructions. β-actin was probed as an internal control. All experiments were repeated three times, and similar results were obtained.