Rabbit anti stat1

The cells were harvested and washed three times with PBS and then lysed in radioimmune precipitation assay (RIPA) buffer (Beyotime, China) with PMSF (Biosharp, China). The mixture was centrifuged at 15,000 g for 15 min, and the supernatant was collected. Protein concentration was determined by BCA Protein Assay Kit (Beyotime, China). Twenty five microgram g total protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, United States), and then the membranes were incubated with 5% bovine serum albumin (BSA) with primary antibodies. The primary antibodies used are as follows: rabbit anti-DENV-2 Capsid (Gentex, United States), rabbit anti-STAT1, rabbit anti-p-STAT1 phosphorylated Tyr701, rabbit anti-STAT2, rabbit anti-p-STAT2 phosphorylated Tyr690, rabbit anti-USP18 (Cell Signaling Technology, United States), mouse anti-GAPDH (Zengneng, China). The secondary antibodies included HRP-conjugated ECL goat anti-rabbit IgG or HRP conjugated ECL goat anti-mouse IgG (Beyotime, China). The protein bands were exposed using the ECL Western Blotting Analysis System (Millipore, United States) on ImageQuant LAS 4000 mini (GE, United States). Densitometry was performed with ImageJ software.

The following primary antibodies were used for Western blotting: rabbit anti-pPKD1 S744/S748 (pPKD1/2 activation loop), rabbit anti-pPKD1 S916, rabbit anti-PKD1, rabbit anti-PKD2, anti-pSTAT1 Y701, rabbit anti-STAT1 (Cell Signaling), rabbit anti-lamin B1 (LB1) (Proteintech), and rabbit anti-pPKD2 S876 (Millipore). The rabbit antibody to HRV 2C was generated and used as previously described (9 (link)). Secondary antibodies conjugated to horseradish peroxidase (HRP) were obtained from Jackson ImmunoResearch and were revealed by using the ECL reagent (Geneflow). PDBu) (Sigma) was dissolved in DMSO and used at a final concentration of 200 nM in all the experiments as a positive control for PKD phosphorylation. For the interferon receptor (IFNAR)-blocking antibody studies, either a mouse anti-IFNAR2 antibody (Stratech) or isotype control mouse IgG2a (Abcam) was used at a 5-μg/ml final concentration. Recombinant human IFN-β1α (IFN-β; R&D) was used at a final concentration of 30 U/ml. The primary antibodies used for confocal microscopy were mouse anti-Myc (Merck Millipore) and rabbit anti-GM130 (BD Pharmingen) in combination with a donkey anti-rabbit secondary antibody coupled to Alexa 546 and a donkey anti-mouse secondary antibody coupled to Alexa 488, respectively (Jackson ImmunoResearch).

Human trophoblast stem cell 1,049 cells were differentiated into EVT in 10 cm dishes for 5 days. Cell lysate was collected every day for 5 days using RIPA buffer (Fisher Scientific, United States) containing protease and phosphatase inhibitors (Roche Applied Science, United States) according to the manufacturer’s protocol. Protein concentration was quantified by BCA protein assay (Thermo Scientific, United States). Thirty μg of total protein was loaded onto a 10% denaturing polyacrylamide gel for separation and then transferred to PVDF membranes by electrophoresis. Membranes were blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (Sigma-Aldrich) for 1 h and then incubated overnight with primary antibodies: rabbit anti-STAT1 (Cell Signaling Technology or CST #9175) or mouse anti-ACTB (Sigma-Aldrich #A5441). Followed by 1-h incubation with HRP-conjugated secondary antibodies (donkey anti-rabbit IgG, CST #7074S or anti-mouse IgG, CST #7076S), signals were developed using SuperSignal West Dura Extended Duration substrate (Thermo Fisher, United States) and captured on film.