Rabbit anti pjak1

In the present study, the following primary and secondary antibodies were used: rabbit anti-pJAK1 (Cell Signaling, Beverly, MA), rabbit anti-pSTAT3 (Cell Signaling), rabbit anti-IL-6 (Cell Signaling). For immunohistochemistry, endogenous peroxidases were quenched in 0.3% H₂O₂ in water for 10 min and then blocked with 10% normal goat serum in PBST. For detection of intracellular antigens, tissues were incubated with 100% methanol -20°C and permeabilized in blocking solution with 0.3% Triton X-100. The primary antibody in blocking buffer was applied to tissues for 60 min at room temperature or overnight at 4°C. Sections were washed in PBS and then incubated with goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA) or anti-rat biotinylated IgG (BD Biosciences, San Jose, CA). Staining was detected using Vectastain ABC kit (Vector Labs, Burlingame, CA) and sections were counterstained with hematoxylin. The area of positive area and integral optical density was calculated on the Image-pro Plus 6.0.

Cells plated on 22-mm, 1.5-in thick poly-l-lysine-coated German coverslips (Neuvitro, Cat #GG-22-1.5-PLL) were fixed with 4% paraformaldehyde (Santa Cruz, Cat #30525-89-4), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Cat #9002-93-1), and blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature on a shaker. Followed by overnight incubation with rabbit anti-human MUC1-CT (Thermo Fisher Scientific), the cells were incubated with goat anti-rabbit IgG conjugated with Alexa Fluor 555 for 45 min at 37 °C. Cells were then counterstained with DAPI. For dual staining of p-JAK1 and MUC1-CT, the cells were co-incubated with Armenian hamster anti-MUC1-CT (Thermo Fisher Scientific) and rabbit anti-p-JAK1 (Cell Signalling) overnight at 4 °C. Followed by incubation with goat anti-Armenian hamster IgG conjugated with Alexa Fluor 555 (Thermo Fisher Scientific) and goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Cell Signaling) at room temperature for 45 mins, the cells were counterstained with DAPI. Images were obtained with a Zeiss 710 confocal microscope.

The quantification of protein was performed in HepG2.2.15 cells using Western blot analysis, as previously reported [20 (link)]. Rabbit anti-ISG15, rabbit anti-pSTAT1, rabbit anti-STAT1, rabbit anti-pJAK1 and rabbit anti-JAK1 were purchased from cell signaling technology (CST, Danvers, MA), rabbit anti-MxA was purchased from Genetex (Irvine, USA), rabbit anti-IFIT1 was purchased from Bioss (Beijing, China), rabbit anti-USP18 and mouse monoclonal anti-HBcAg were purchased from Abcam (Cambridge, UK), rabbit anti-GAPDH was purchased from Protein Tech Group (Wuhan, China). Secondary antibodies were added for signal detection. An enhanced ECL chemiluminescence detection kit (Millipore, Billerica, MA) was used for detection. The quantification of protein bands related to GAPDH was performed by analysis with FUSION FX imaging system (Vilber, French).