Hrp linked anti mouse igg antibody

Cells were seeded in six-well plates at a concentration of 5 × 10⁵ per well in 1 mL of medium and transfected with miRNA mimics or miRNA inhibitors. The cells were stimulated with type I IFN (1000 U/mL) 24 h after transfection and the cells were lysed at the indicated time points. The proteins were extracted, separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and probed with the specified primary antibodies directed against phosphorylated STAT1 (p-STAT1), STAT1, p-STAT2, STAT2, JAK1, PTP1B, p-P44/42 MAPK (ERK1/2), P44/42 MAPK (ERK1/2), p38 MAPK, p-p38 MAPK, and p-TYK2 (Cell Signaling Technology, diluted 1:1000); anti-STAT3, anti-p-STAT3, and anti-p-JAK1 (Santa Cruz Biotechnology, diluted 1:200); anti-TYK2 and anti-β-tubulin (Abcam, diluted 1:1000 and 1:5000, respectively). The secondary antibodies used were obtained from Cell Signaling: horseradish-peroxidase (HRP)-linked anti-rabbit IgG antibody (diluted 1:5000), and HRP-linked anti-mouse IgG antibody (diluted 1:4000). The signal was generated with SuperSignal® West Pico Luminol/Enhancer Solution (Pierce). We used Tanon 6600 Luminescent Imaging Workstation (Tanon Science & Technology, Shanghai, China) to scan the film. And band signal intensity was analyzed using Image J.

Protein (20–50 μg) was extracted from the ipsilateral spinal cord dorsal horn and separated with 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and incubated with antibody against CXCR4 (1:1000; ab124824, Abcam), VDUP-1 (1:1000; sc-271238, Santa Cruz Biotechnology), NLRP3 (1:500; NBP1-77080, Novus Biologicals), apoptosis-associated speck-like molecule containing CARD domain (ASC; 1:1000; sc-22514-R, Santa Cruz Biotechnology), Caspase1 (1:1000; ab-1872, Abcam), Caspase1 p10 (1:1000; sc-514, Santa Cruz Biotechnology), and IL-1β (1:1000; ab9722, Abcam) or control β-Actin (1:5000; YM3028, ImmunoWay). Blots were washed and incubated in HRP-linked anti-rabbit IgG antibody (1:5000; 7074, Cell Signaling Technology) and HRP-linked anti-mouse IgG antibody (1:5000; 7076, Cell Signaling Technology). Protein blots were visualized using Clarity ECL Substrates (Biorad).

After the indicated treatment, HAECs were lysed using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) containing protease and phosphatase inhibitors (Sigma-Aldrich, USA). Cytoplasm was removed using hypotonic buffer to obtain the nuclear extracts via lysis. Then, 20 μg cell lysates was immobilized using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein mix was then transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA) to separate the proteins according to size. The membranes were then blotted against their specific antibodies and corresponding secondary antibodies. The immunoblot bands were visualized using Pierce™ ECL Plus western blotting substrate (Catalog # 32132) [33 (link)]. The following antibodies were used: p-JNK (1:1000, # 9255, Cell Signaling Technology, USA); JNK (1:3000, #9252, Cell Signaling Technology, USA); LOX-1 (1:2000, ab214427, Abcam); KLF6 (1:1000, #AF3499, R&D Systems); β-actin (1:5000, #4970, Cell signaling technology, USA); HRP-linked anti-rabbit IgG antibody (1:2000, #7074, Cell signaling technology, USA); HRP-linked anti-mouse IgG antibody (1:2000, #7076, Cell Signaling Technology, USA).