Hrp conjugated goat anti mouse igg antibody

The infected cells were lysed using NP-40 lysis buffer (containing 1% NP-40, 50 mM Tris, 150 mM NaCl, 5 mM EDTA, and protease inhibitor cocktails (Himedia, Mumbai, India)) and centrifuged at 12,000× g for 20 min to obtain the cell lysate. For DENV2 protein detection, cell lysates were subjected to SDS-PAGE under nonreducing conditions. The 4G2 monoclonal antibody (HB-112, ATCC, Manassas, VA, USA) was used as the primary antibody for the DENV2 E protein. Mouse anti-β-actin antibody (Biolegend, San Diago, CA, USA) was used as a loading control. HRP-conjugated goat anti-mouse IgG antibody was used as a secondary antibody (Biolegend, San Diago, CA, USA). The chemiluminescence signals were developed using Immobilon Classico Western HRP substrate (Merck, Darmstadt, Germany). Protein band intensities were quantified using ImageJ software version 1.53.

For membrane markers, 40
μL of sEVs
(1 × 10¹⁰ particles/mL) and for intraluminal and negative
markers, 1 × 10¹⁰ particles in 40 μL were spotted
on nitrocellulose membranes (0.45 μm; Bio-Rad) and incubated
at RT for 1 h in blocking buffer (3% milk in TBS-T). Mouse anti-human
CD63 (BioLegend #353013), CD81 (BioLegend #349520), CD9 (BioLegend
#312102), and Calnexin (GeneTex #GTX629976-S) antibodies at 0.5 μg/mL
and Alix (Cell Signalling Technology #2171S) at 0.2 μg/mL in
blocking buffer were added to separate membranes and incubated overnight
at 4 °C. Staining was performed with an HRP-conjugated goat anti-mouse
IgG antibody (1:10,000 dilution in blocking buffer; BioLegend #405306)
for 1 h at RT. A chemiluminescence signal was detected using a SuperSignal
West Atto Ultimate Sensitivity substrate (Thermo Fisher), imaged on
iBright FL1000 (Invitrogen) or developed on a CL-Exposure film (Thermo
Fisher).