Precast gel

A standard protocol was adapted for the Western blot assay. Briefly, the same pfu of virus supernatant was loaded into separate wells of the precast gel (BioRad #456–1084). We then probed the membrane for specific viral proteins E (clone 4G2, CTK B7052 USA), prM (Genetex gtx108092), NS1 (Genetex gtx103346) and Capsid (A gift from Dr. John G Aaskov, Clone 6F3–1).

Purified EVs or cell lysates were added to reducing buffer in 1 : 1 ratio and boiled for 10 min at 95°C. Samples were loaded in a 4-20% 1.5 mm Bio-Rad precast gel and allowed to migrate at 100 V. Proteins were transferred to a nitrocellulose membrane in a transfer chamber at 45 mA overnight. The membrane was blocked in 5% nonfat dry milk prepared in 0.2% Tween-20 and 1× TBS for 30-45 min at RT. Primary antibodies CD9 (1 : 250), Hsp70 (1 : 250), NF-κB (1 : 250), and IRF-8 (1 : 250) (DSHB, Iowa City, IA, USA) were used in probing the membranes overnight at 4°C. Nitrocellulose blots were washed three times in wash buffer (0.2% Tween-20 in 1× TBS) for 10 min and incubated with HRP-conjugated secondary antibody (1 : 500-1 : 2000) diluted in blocking solution for 1 h at RT with gentle shaking. The membrane was washed three times in wash buffer for 10 min and developed using SuperSignal West Femto Maximum Sensitivity Substrate (vendor). The signal was detected using Bio-Rad ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA) [24 (link)].

For immunoprecipitations, 6x10¹⁰ of MCF-7 and HCC1937 Cells were harvested and lysed in lysis buffer (20 mM NaCl, 30 mM KCl, 0.1% DTT, 0.5% Triton X-100, supplemented with protease and phosphatase inhibitor cocktail, Halt Protease Inhibitor Cocktail/ Halt Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific Inc.); cell lysates were clarified by centrifugation at 18,000xg for 30 min. BRCA1 was immunoprecipitated with 10 ng of anti-BRCA1 monoclonal antibody (clone D-20, Santa Cruz) during an overnight incubation with 1 ml of total cells extract (0.5 μg/μl). 20 μl of resuspended volume of Protein A/G PLUS-Agarose (Santa Cruz) were added to the mixture and incubated at 4° C on rotating device for 2 hour. Immune complexes were collected by low speed centrifugation, washed three times in 1 ml lysis buffer, and boiled in 20 μl of SDS loading buffer; denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (4–15% Bio-Rad precast gel). Proteins were transferred to Nitrocellulose membrane, which was blocked in 5% nonfat milk, 150 mM NaCl, 10 mM Tris (pH 8.0), and 0.05% Tween. Immunoblots were performed with rabbit anti-ATF1 antisera at 1 mg/ml (C-20 Santa Cruz.) and developed by enhanced chemiluminescence (Santa Cruz).