Anti rabbit hrp linked antibody

Cells were seeded (2 × 10⁵) in a six well plate for 24 h and were treated with GE at 150 and 200 (µg/mL) for 24 h followed by washing with pre-cooled PBS. Ice cold RIPA buffer with protease/phosphatase was added for the cell lysis. The lysates were collected and centrifuged at 13,000× g for 30 min at 4 °C. The total protein concentration in cell lysates was measured by the Bradford method. The cell extracts were separated by SDS polyacrylamide gels (8–10%), and then, the protein was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk, incubated with indicated primary and appropriate secondary antibodies (anti-rabbit IgG-HRP conjugates), and protein bands were detected using 1-Step Ultra TMB-Blotting Solution (Thermo Scientific). STAT1 (D1K9Y) Rabbit mAb, Phospho-STAT1 (Tyr701) (58D6) Rabbit mAb, STAT2 (D9J7L) Rabbit mAb, Phospho-STAT2 (Tyr690) (D3P2P) Rabbit mAb, JAK1 (6G4) Rabbit mAb, Phospho-JAK1(Tyr1034/1035) (D7N4Z) Rabbit mAb, TYK2 (D4I5T) Rabbit mAb, Phospho-TYK2 (Tyr1054/1055) (D7T8A) Rabbit mAb, β-Actin (13E5) Rabbit mAb and Anti-rabbit HRP-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Densitometric analysis was performed by Image J software version 1.44.

Left ventricular tissues were lysed in RIPA buffer containing phenylmethanesulfonyl fluoride (PMSF). Protein concentration was measured by using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Protein samples (30 μg) were separated by 8% or 10% SDS-PAGE, followed by transferring to the PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk diluted in 1x Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h, the blotted membrane was incubated with corresponding primary antibodies (diluted with 5% BSA in TBST) at 4°C overnight, including anti-fibronectin (FN, Boster Biological Technology, Wuhan, China), anti-transforming growth factor-β (TGF-β, Abcam, USA), anti-collagen I (Bioworld Technology, USA), anti-β-tubulin (Cell Signaling Technology, USA), and anti-α-smooth muscle actin (α-SMA, Bioworld Technology, USA). Then, corresponding secondary anti-rabbit HRP-linked antibodies (Cell Signaling Technology, USA) were incubated with the membrane at room temperature for one hour. Blotting bands were visualized using an enhanced chemiluminescence reagent kit (Cell Signaling Technology, USA), and the gray values were analyzed using the Quantity One software (Bio-Rad, USA).

Protein samples in appropriate buffers according to the experiments were diluted in Laemmli buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 0.05% bromophenol blue, and pH 6.8) and heated 10 min at 100°C. After electrophoresis in a 10% acrylamide SDS-PAGE gel, proteins were transferred onto a nitrocellulose membrane at 100 V for 1 h in a Tris/glycine buffer supplemented with 10% ethanol. After blocking of the nitrocellulose membrane with 0.05% TBS Tween-20 (TBS-T) supplemented with 5% milk for 1 h at room temperature, membrane was probed with primary antibodies diluted at 1/1,000 in TBS-T with 3% BSA overnight at 4°C. Membrane was then washed in TBS-T and incubated with HRP-linked secondary antibodies diluted at 1/10,000 in TBS-T with 5% milk at room temperature. Anti-rabbit HRP-linked antibodies and anti-mouse HRP-linked antibodies (Cell Signaling) were used for protein detections. Chemiluminescent protein detection was done using ECL Prime and ODYSSEY Fc (Lycor) hardware. Rabbit monoclonal HA-Tag antibodies (C29F4) used for Western blotting were purchased from Cell Signaling.