Ecl plus kit

Cells or skin samples crushed in liquid nitrogen were solubilized at 4°C in lysis buffer (0.5% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 100 μg/ml phenylmethylsulphonyl fluoride, and protease inhibitor cocktail). Five micrograms of protein were separated on SDS-polyacrylamide gels and transferred onto polyvinylidine fluoride membranes (Bio-Rad, Hercules, CA USA). Non-specific antibody binding was blocked by incubating the membranes in 5% w/v non-fat milk powder in TBS-T (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% v/v Tween-20). The membranes were incubated with rabbit anti-POSTN antibody (Abcam, Cambridge, UK) diluted 1:1000 overnight at 4°C or with mouse anti-β-actin antibody (Sigma- Aldrich, St Louis, MO, USA) diluted 1:10,000 overnight at 4°C. Then, the membranes were washed 3 times in TBS-T for 5 min. Finally, the membranes were incubated with either HRP-conjugated anti-mouse or anti-rabbit at a dilution of 1:10,000 for 60 min at room temperature. Protein bands were detected using the ECL Plus kit (Thermo Scientific, Rockford, IL USA). The intensity of the bands was quantified using NIH image J software.

293T cells were lysed in radioimmunoprecipitation assay (RIPA) buffer after transfection with shRNA for 72 h. A BCA Protein Assay Kit was used to detect the total protein concentration. Equal amounts of protein lysates were electrophoresed in 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked by Tris-buffered saline with Tween (TBST) containing 5% milk. Protein expression was probed with mouse anti-FLAG (F1804; Sigma-Aldrich; Merck KGaA) and goat anti-mouse IgG (SC-2005; Santa Cruz Biotechnology, Inc.). Protein expression in 293T cells that were exogenously transfected with a plasmid encoding FLAG-tagged ZWINT was detected by mouse anti-FLAG and mouse anti-GAPDH (SC-32233; Santa Cruz Biotechnology, Inc.). The ECL-PLUS kit (Thermo Fisher Scientific. Inc) was used to detect the signals on X-ray film. GAPDH was used as the reference protein to calculate the relative protein levels. Independent tests were performed in triplicate.

Total protein was extracted using RIPA lysis buffer with PMSF (Beyotime). After the concentration of protein was detected using a BCA protein Assay Kit (Generay, Shanghai, China), the same amount of protein (30 μg/lane) was subjected to SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with non-fat milk for 2 h at room temperature and were incubated with the primary antibodies at 4°C overnight. On the next day, membranes were incubated with secondary antibody solution (goat anti-mouse IgG). Chemiluminescent signals were detected using an ECL plus kit (Thermo Fisher Scientific), and were quantified with Image J software.

Western blotting was performed with angiogenesis markers angiopoietin-1 (1:1000; Millipore, Billerica, MA, USA) and Ang-2 (1:1000; Abcam, Cambridge, MA, USA), endothelial nitric oxide synthase (eNOS) (1:1000; Cell Signaling, Beverly, MA, USA) and phosphorylated-eNOS (Cell Signaling; 1:1000) as described previously.^{24 (link)} The anti-β-actin antibody (1:10,000; Sigma) was used as control. Homogenates of the entire ischemic as well as contralateral (non-ischemic) hemispheres were obtained. Equal amounts of protein (80 μg) for each group were assayed and proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. Membranes were blocked with 5% (w/v) bovine serum albumin in Tris-Buffered Saline with Tween 20 for 1 hour. After immunoblotting with specific antibodies, membranes were incubated with HRP-conjugated IgG (1:1000; GE Healthcare, Pittsburgh, PA, USA), and labeled proteins were detected using the electrochemiluminescence (ECL) Plus kit (Thermo Scientific, West Palm Beach, FL, USA). The band intensities obtained by western blot analysis were determined using the ImageJ program. All samples were analyzed at least in triplicates.

MNNG/HOS and U2OS cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology) on ice for 30 min. The lysate was centrifuged at 15,000 × g, 4°C for 15 min, and the supernatant was heated at 100°C for 5 min. The concentration of the protein samples was determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Following separation by 10% SDS-PAGE (20 µg protein/lane), the proteins were transferred onto polyvinylidene fluoride membranes (MilliporeSigma). Subsequently, the membranes were blocked at room temperature with 5% skim milk for 1 h and incubated with primary antibodies against IARS2 (1:2,000; MilliporeSigma; SAB4502342), cleaved-caspase-3 (1:500; Cell Signaling Technology, Inc; cat. no. # 9664S), BAX (1:500; Abcam; ab32503) or β-actin (1:5,000; Abcam; ab8226) at 4°C overnight. Subsequently, the membranes were washed with Tris-buffered saline-0.5% Tween and incubated at room temperature with goat anti-mouse IgG-HRP secondary antibody (1:2,000; Santa Cruz Biotechnology, Inc; cat. no. #7076) for 2 h. Protein bands on the membranes were then detected using an ECL-PLUS/Kit (Thermo Fisher Scientific, Inc.).