Rabbit anti phospho c jun

For immunostaining, tissues were fixed in Zn-formalin for 24 hr and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 min each, and then blocked in 5% donkey serum for 1 hr at room temperature, incubated with either Rabbit anti-BNIP3 antibody at 1:100 (A5683, Abclonal), Rabbit anti-NF-kB-p65 1:400 (8242S, Cell Signaling Technology), Rabbit anti-Phospho-c-jun 1:200 (3270S, Cell Signaling Technology), Rat anti-ki67 1:100 (14-5698-82, ebiosciences), Rabbit anti-pRB-Ser807/811 1:400 (8516, Cell Signaling Technology), anti-p21 1:1000 (ZRB1141, Sigma), Rat anti-Phospho H3 1:1000 (H9908, Sigma), or Goat anti-GFP (ab6673, Abcam) at 1:250 overnight at 4°C, washed, incubated with secondary antibody AF594-anti-rabbit (A-21207, Invitrogen), Alexa594 anti-rat (A-21209, Invitrogen), and Alexa488 anti-goat (11055, Invitrogen) antibodies at 1:200 for 1 hr at room temperature, counterstained with DAPI, washed, and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera using ×400 magnification. MFI was calculated by measuring average intensity over a fixed threshold for all images. NF-kB staining was imaged via Zeiss LSM880 confocal microscope using ×63 oil-immersion objective, and intensity was measured on 0.8 µm z-sections.

Aze was purchased from Selleckchem (Houston, TX, USA). SP600125 was obtained from Calbiochem (Darmstadt, Germany). LPS (Escherichia coli, serotype 011:B4), mouse anti-β-actin (1:2000, cat# A5316) and rabbit anti-lamin B1 (1:2000, cat# SAB1306342) antibodies, dimethyl sulfoxide (DMSO), and adenylyl-imidodiphosphate (AMPPNP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor 488-conjugated anti-mouse immunoglobulin G (IgG) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Thermo Scientific (Rockford, IL, USA). Antibiotic-antimycotic reagent, Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). The rabbit anti-phospho-IκBα (1:1000, cat# ab97783) antibody was supplied by Abcam (Cambridge, MA, USA). Rabbit anti-phospho-JNK (1:1000, cat# 4668), rabbit anti-JNK3 (1:1000, cat# 2305), rabbit anti-phospho-c-Jun (1:1000, cat# 9261), rabbit anti-c-Jun (1:1000, cat# 9165), rabbit anti-iNOS (1:1000, cat# 2982), rabbit anti-COX2 (1:1000, cat# 4842), mouse anti-NF-κB p65 (1:1000, cat# 6956), mouse anti-IκBα (1:1000, cat# 4814) antibodies, and horseradish peroxidase-conjugated anti-rabbit (1:2000, cat# 7074) and anti-mouse IgG (1:2000, cat# 7076) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Immunohistochemistry staining for STAT1, IRF1, IRF8 was conducted as described previously (15 (link)). Briefly, skin sections were incubated with antibodies: Rabbit anti-phospho-c-Jun (1:200, Cell signaling, catalog 3270), Rabbit anti-FosB (1:50, Cell signaling, catalog 2251), Rabbit anti-phospho-STAT1 (1: 800, Cell signaling, catalog 9167), Rabbit anti-IRF1 (1:400, Cell signaling, catalog 8478), Rabbit anti-IRF8 (1:2000, Abcam, catalog ab207418). As negative controls, Rabbit IgG isotype control (1: 400, Cell signaling, catalog 3900) was used (data shown in Supplementary Figure 5). Images were obtained from three typical areas for each sample. The quantification of nuclear localization of each protein was evaluated with ImageJ.