Anti cav 1

PBMCs were collected by cytospin and stained using the indicated primary antibodies. Cells were fixed with 2% paraformaldehyde and then permeabilized with 0.1% Triton X-100. Cells were incubated with Alexa Flour^® 647-conjugated anti-CD3, CD14, or CD19 (BioLegend, San Diego, CA), and anti-CAV-1 (Santa Cruz Biotechnology), followed by Alexa Flour^® 488-conjugated secondary IgG (Invitrogen, Grand Island, NY). Appropriate isotype IgG was used as a control. ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole (DAPI; Life Technology, Carlsbad, CA) was used for nuclear identification and mounting. The intensity of CAV-1 staining was quantified in randomly chosen CD3⁺ T cells, CD19⁺ B cells, and CD14⁺ monocytes. The data were obtained from 20 cells from each three patients and healthy subjects. Images were taken and analysed using an Olympus Fluoview 1000 microscope (Olympus America, Melville, NY) and fixed camera settings.

As described previously (Reppetti et al., 2019 (link)), control cells and hyperosmolarity-treated cells were collected in a lysis buffer. Then, protein concentration was determined using the commercial Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States).
For immunoblotting studies, 50 μg proteins from each sample of the lysate of Swan 71 cells were loaded and resolved on a 12.5% polyacrylamide gel and onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia Biotech Ltd., Pittsburgh, PA, United States). After blocking, membranes were rinsed and incubated overnight with an anti-Cav-1 (1:1,000; Santa Cruz Biotechnology Incorporation, CA, United States) followed by incubation with a peroxidase-conjugated secondary antibody (1:10,000; Jackson Immuno Research Laboratories, Incorporation, West Grove, PA, United States). Immunoreactivity was detected using the enhanced chemiluminescence (ECL) detection reagent (Amersham ECL plus, GE Healthcare Life Sciences, Pittsburgh, PA, United States). Bands were quantified by densitometric analysis using the ImageJ 1.51r^® software package (Bethesda, MD, United States). Results were expressed as the relative abundance of each protein and we used Ponceau staining as a loading control (Gilda and Gomes, 2013 (link)).

Cells were lysed with radioimmunoprecipitation (RIPA) assay buffer containing 1× PBS, 1% (v/v) Nonidet P-40 (NP-40), 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.1 mg/mL phenylmethylsulfonyl fluoride, 30 μL/mL aprotinin, and 1 mM sodium orthovanadate. Cell lysates were centrifuged and the resulting supernatants were collected. Proteins were separated by 8%–15% SDS–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. Each membrane was blocked in Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% non-fat dry milk for 1 h at room temperature, followed by an overnight incubation with a primary antibody in TBST containing 1% non-fat dry milk at 4 °C. Anti-cleaved caspase-3, anti-cleaved poly (ADP-ribose) polymerase (PARP), anti-B-cell lymphoma 2 (Bcl2), anti-ATPase inhibitory factor (ATPIF)-1, anti-HO-1, anti-p62, anti-CAV1, anti-collagen, and anti-glyceraldehyde phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were washed with TBST and incubated with goat anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG secondary antibody for 2 h. Signal was measured using the chemiluminescence system (GE Healthcare, Piscataway, NJ, USA).

The mouse monoclonal antibody directed against NS3 (D-7) and the rat monoclonal antibody against NS5 (13G7) have been previously described [44] (link), [45] (link).Rabbit polyclonal anti-Cav-1 and mouse monoclonal anti-Flt-2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rat polyclonal antibodies targeting NS2B and NS3 were produced in our laboratory. Mouse monoclonal anti-TfR antibodies were obtained from Zymed (San Francisco, CA, USA). Mouse monoclonal anti-dsRNA antibodies were obtained from English & Scientific Consulting (The data obtained in the present study demonstrated that DENV proteins colocalized with lipid raft-resident proteins. Cholesterol is an important component of lipid rafts. Moreover, previous research has demonstrated the dependence of DENV entrance on cholesterol and a likely role for cholesterol in viral replication [26] (link). Fluorescein isothiocyanate goat (1∶100) and Texas Red-labeled goat anti-mouse antibodies were also used.