Anti rabbit hrp conjugated secondary antibody

Western blot analysis was performed as previously described[16 (link)]. Briefly, after previous treatment, HUVECs were harvested and homogenized in lysis buffer. Total protein was separated by SDS-PAGE and transferred to PVDF membranes (Millipore Corporation, USA), which were blocked with 5% non-fat milk in Tris-buffered saline and 0.1% Tween 20 for 2 h, followed by incubation with primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Finally, the protein bands of interest were visualized using an enhanced chemiluminescence (ECL) detection reagent (General Electric Healthcare company, USA) and were detected using a fluorescence imaging analysis system (ImageQuant^TM LAS 4000 mini, General Electric Healthcare company, USA). The primary antibodies for BCL-2, BAX, CCND-2, caspase-3, and α-Tubulin were all purchased from Cell Signaling Technology. A HRP-conjugated anti-rabbit secondary antibody was purchased from General Electric Healthcare Company. The protein levels were normalized to α-tubulin.

Aortic tissue homogenates were dissolved in sample buffer, run on a NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen, CA, United States), and transferred to nitrocellulose membranes using the iBlot transfer system (Invitrogen). Membranes were washed in PBS and blocked for 1 hr at room temperature with 5% instant non-fat dry milk dissolved in PBS containing 1% Tween-20 (Sigma, MO, United States) (PBS-T). Equal protein loading of samples was determined by a protein assay (Bio-Rad, CA, United States) and confirmed by probing with antibodies against β-Actin (Sigma). Membranes were probed overnight at 4° centigrade with primary antibodies against pSmad3 (1880-1; Millipore), Smad3 (#9513; Cell Signaling), pERK1/2 (#4370; Cell Signaling), ERK1/2 (#4695; Cell Signaling), pPKCβ (#75837; Abcam, United Kingdom), PKCβ (#32026; Abcam), pPLCγ (#2821; Cell Signaling), and PLCγ (#2822; Cell Signaling), dissolved in PBS-T containing 5% milk. Blots were then washed in PBS-T and probed with HRP-conjugated anti-rabbit secondary antibody (GE Healthcare, United Kingdom) dissolved in PBS-T containing 5% milk at room temperature. Blots were then washed in PBS-T, developed using SuperSignalWest HRP substrate (Pierce Scientific, IL, United States), exposed to BioMax Scientific Imaging Film (Sigma), and quantified using ImageJ analysis software (NIH, MD, United States).

Qualitative analysis of acetylated proteoforms was conducted on 2-D DIGE total protein extracts. 2-D immunoblotting was carried out on DMD, BMD1 and BMD2 pooled samples by subjecting each pool (150 μg) to isoelectrofocusing on 18 cm, 6–10 pH-gradient IPG strips (GE Healthcare), with a voltage gradient ranging from 300 to 8000 V, for a total of 52,000 Vh, using an IPGphor electrophoresis unit (GE Healthcare). After focusing, proteins were reduced and alkylated. The second dimension was carried out in 20 × 25 cm², 12% polyacrylamide gels at 20 °C. Blots were blocked in 5% BSA for 30 min and incubated with a 1:1 mixture of rabbit anti-acetylated-lysine (Ac-K2–100) (1:1000, Cell Signaling Technology, Danvers, MA, USA, #9814) and anti-acetylated-lysine (1:1000, Cell Signaling Technology, #9441) primary antibodies. After washing, membranes were incubated with anti-rabbit HRP-conjugated secondary antibody (1:10,000, GE Healthcare). Signals were visualized by chemiluminescence using the ECL Prime (GE Healthcare) detection kit and the Image Quant LAS 4000 (GE Healthcare) analysis system.

Protein was extracted from the Tibialis anterior (TA) muscle by homogenization in extraction buffer containing 150mM NaCl, 50mM HEPES, 2.5mM EDTA, 10% Glycerol, 1% Triton, and protease and phosphatase inhibitor cocktails (Roche, UK). The homogenate was centrifuged at ~15,000g for 10 minutes and the supernatent transferred to an equal volume of 2x Laemmli loading buffer. Constant protein was loaded and separated on a 4–15% precast acrylamide gel (Criterion TGX, BioRad) and wet transferred to nitrocellulose membranes, with ponceau staining used to confirm the equal loading of protein. Membranes were then probed with an anti-PEPCK-M antibody (Cell Signaling #6924), followed by an anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Life Science). Bands were visualized using ECL detection reagent (GE Healthcare Life Science) and exposure to photographic film in a dark room. Densitometry of band intensity was conducted to determine protein expression levels.

Western blot analysis was carried out to assess protein expression levels. Total protein was extracted from the cells by addition of lysis buffer (10 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM EDTA) containing protease inhibitor cocktail (Roche, Switzerland) and phosphatase inhibitor cocktail (Roche). Cultured cells were scraped and whole-cell lysates were prepared by centrifugation at 16,363 × g for 15 min at 4 °C. Equal amounts of protein were loaded alongside a pre-stained protein ladder (1st Base) and electrophoresed on 10% SDS-polyacrylamide gels. The resolved proteins were transferred onto PVDF membranes (Immobilon transfer membrane, Millipore Corporation). After electroblotting, the membranes were blocked with Tris-buffered saline with Tween 20 (1xTBST: 20 mM Tris base, 137 mM NaCl, 0.1% Tween 20) containing 5% non-fat dry milk at room temperature for 1 h before overnight hybridization with appropriate concentrations of primary antibodies (Supplementary Table 3) diluted in blocking buffer. The membranes were then incubated with anti-rabbit HRP-conjugated secondary antibody (NA934, GE Healthcare, UK) for 1 h at room temperature. Antigen-antibody complexed to the blotted proteins were detected using chemiluminescence detection system. The chemiluminescent signal was developed on Amersham Hyperfilm ECL (GE Healthcare).