Anti psma2

Cells were grown on coverslips (Thermo Fisher Scientific, Rochester, NY, USA) and washed, fixed in 4% paraformaldehyde for 15 min. at 4°C, and blocked in 5% normal gelatin diluted in PBS with 0.3% Triton X-100. After a brief wash in PBS, cells were incubated with primary antibodies (anti-Golgi 58k, 1:100, Sigma-Aldrich, St Louis, MO, USA; anti-PSMA2, 1:100, Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. The secondary antibodies conjugated with DyLight (KPL, Gaithersburg, MD, USA) were used to detect primary antibodies. Organelle Lights endosomes-RFP (Invitrogen) was employed to visualize endosome of Cx31.1-EGFP-H1299 cells according to the manual of the manufacture. To visualize the colocalization of mCherry-LC3 and Cx31.1-EGFP, Cx31.1-EGFP-H1299 cells were transiently transfected with mCherry-LC3. The cells were then observed under a Nikon ECLIPSE research microscope (Nikon, Tokyo, Japan).
As to the immunofluorescence of clathrin, we fixed the cells in methanol at 4°C for 10 min., and the anti-clathrin heavy chain (1:100, Cell Signaling Technology) was used for detection. Fluorescence signals were observed under the ZEISS LSM710 (Zeiss, Oberkochen, Germany) spectral confocal microscopy.

The protein concentrations in the cell lysates were measured using the Bradford Protein Assay, and 20 µg of protein were resolved in 10% SDS-PAGE gels and transferred to 0.2 µm nitrocellulose membranes. Anti-PSMA2 (Cell Signaling, Danvers, MA, USA, Cat. 2455), anti-ZIKV NS1 (BioFront Technologies, Tallahassee, FL, USA, Cat. BF-1225-06), anti-ZIKV NS3 (Genetex, Irvine, CA, USA, Cat No. GTX133309), anti-ZIKV Env (Genetex, Cat No. GTX133314), anti-STAT1 (Cell Signaling, Cat. 9176S), and anti-Beta-Actin (Cell Signaling, Cat. 3700S) antibodies were used to detect protein targets. Anti-rabbit (Cell Signaling, Cat. #7074) or anti-mouse (Cell Signaling, Cat. #7076) secondary antibody was used to identify the primary antibody conjugates to the targeted proteins. After overlaying with ECL reagents, protein bands were photographed with an Amersham Imager 680 (Gelifesciences, MA, USA). To quantify band intensities, Image J version. 1.53e (NIH, Bethesda, MD, USA) was used, and Graphpad Prism version 6.0. (La Jolla, CA, USA) was used to visualize them graphically.

Western blot was used to determine viral and host cellular protein expression, following the protocol reported previously [20 (link)]. Thirty ug of proteins from different conditions were resolved in 10 or 12% SDS-PAGE gels, then transferred to 0.2 µm nitrocellulose membranes and probed for specific proteins with the following antibodies: anti-PSMA2 (Cell Signaling, Danvers, MA, USA, Cat. 2455), anti-STAT3 (Cell Signaling; Cat #no. 9139S), anti-STAT1 (Cell Signaling, Cat. 9176S), anti-GAPDH (Cell Signaling, Cat.2118L;), anti-Beta-Actin (Cell Signaling, Cat. 3700S), CLIC1anti-CLIC1 (EMD Millipore, Darmstadt, Germany, Cat. MABN46), and in-house prepared IAV mouse-anti-NS1 and mouse-anti-NP [26 (link)]. To identify immunological complexes, appropriate secondary horseradish peroxidase (HRP)-conjugated horse anti-mouse or anti-rabbit antibodies (Cell Signaling, cat. 7076, cat. 7074, respectively) were used. Protein bands were visualized with ECL reagents and photographed using an Alpha Innotech FluorChemQ MultiImage III instrument (ALT American Laboratory trading, San Diego, CA, USA). Image J 1.50i was used to measure band intensities to evaluate the differences in protein expression (NIH, Bethesda, Maryland, USA). GraphPad Prism v 9.1.0 (La Jolla, CA, USA) software was used to analyze and visualize the data.