Rabbit anti pstat1

Cells were lysed in LDS sample buffer (Invitrogen), separated by electrophoresis on NuPage 4–12% Bis-Tris gels (Invitrogen) and blotted onto nitrocellulose membranes (GE Healthcare). Membranes were incubated with rabbit anti-HSP90 (Santa Cruz Biotechnology), mouse anti-SAMHD1 (OriGene), rabbit anti-pSAMHD1 (ProSci), mouse anti-cdc2 (Cell Signaling), rabbit anti-CDK1 (proteintech), rabbit anti-CDK2 (proteintech), rabbit anti-CCNE2 (proteintech), rabbit anti-cyclin A2 (proteintech), rabbit anti-STAT1 (proteintech), rabbit anti-pSTAT1 (Cell Signaling), mouse anti-HSV-1 gD (Santa Cruz Biotechnology) or mouse anti-IDO1 (proteintech). Thereafter, membranes were incubated with goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680RD (respectively LI-COR Biosciences), and scanned using a LI-COR Odyssey infrared imaging system. Alternatively, membranes were incubated with appropriate horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmunoResearch), and visualized using SuperSignal West Femto Chemiluminescent solution (Thermo Fisher) and a C-DiGit Western Blot Scanner.

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).

Primary mouse brain vascular pericytes were obtained from iXCells Biotechnologies (10MU-014). All experiments were performed within five passages, wherein cells were grown on collagen-coated (Sigma) chambered slides in pericyte medium containing 2.5% FBS, penicillin-streptomycin, and pericyte growth solution (ScienCell). Once the cells reached ∼70% confluence, they were treated with either DMSO (vehicle) or 100 ng/ml IFN-γ (R&D) for 48 h. Immunocytochemistry was performed by fixing cultures with 4% PFA for 10 min, followed by incubation with the primary antibodies rabbit anti-CC3 (1:100; Cell Signaling), rabbit anti-Pdgfrβ (1:100; Cell Signaling), and rabbit anti-pSTAT1 (1:100; Cell Signaling) for 1 h at room temperature. The samples were incubated with appropriate Alexa Fluor secondary antibody and DAPI and then imaged using a Zeiss 780 LSM confocal microscope. The number of apoptotic pericytes was determined by counting the number of CC3⁺ cells versus the total number of DAPI⁺ cells using Zen software (a minimum of three 20× images were captured and analyzed per experiment; three independent experiments were performed).