Anti mouse igg h l hrp conjugate

To determine cleaved caspase-1 p20 fragment, Western blot assay (WB) was performed. After cell treatment with viral proteins, supernatant was collected and centrifuged at 600×g for 10 min to remove cellular debris. Then, the protein content of the supernatants was concentrated 10× using centrifugal filters with a 10-kDa cutoff (#UFC501096, Amicon, Merck). The concentrated samples were boiled in a reducing sample buffer and separated in 4%–12% polyacrylamide gel (#NW04122BOX, Thermo Fisher Scientific) electrophoresis (PAGE) in MES SDS running buffer (#B0002 Invitrogen, Thermo Fisher Scientific). The proteins from the SDS-PAGE gel were blotted onto a 0.2-µm nitrocellulose (NC) membrane (#LC2000, Invitrogen, Thermo Fisher Scientific) by wet transfer. The membrane was blocked with 5% BSA in PBS for 1 h at RT and rinsed with TBST. The membrane was then incubated with primary antibodies in TBST with 1% BSA overnight at 4°C. The primary antibodies against human caspase-1 (clone Bally-1, #AG-20B-0048-C100, RRID: AB_2490257, Adipogene) were used at 1:1,000 dilution. Thereafter, the membrane was incubated with secondary antibodies Goat Anti-Mouse IgG (H+L)-HRP Conjugate (Bio-Rad) diluted 1:5,000 in TBST with 1% BSA for 1 h at RT. The horseradish peroxidase (HRP) enzymatic reaction was developed using chemiluminescent substrate (#34094, Thermo Fisher Scientific).

Proteins were extracted from EV preparation and tissue by addition of lysis buffer (150 mmol/L sodium chloride, 1.0% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl sulfate), 50 mmol/L Tris) or 10 mmol/L Tris/HCl pH 7.5; 150 mol/L NaCl; 0.5 mmol/L EDTA; 0.5% NP-40) containing proteases and phosphatases inhibitor cocktail (Roche). After clarification (12,000 × g at 4 °C, 20 min), protein concentration was quantified with Bradford assay and the different samples were resuspended in sample buffer. Bio-Rad system was used to run the blots, at constant 120 V, followed by semi-dry transfer at constant 140 mA. After blocking (3% BSA in TBST) the membranes were incubated with primary antibodies overnight (Supplementary Data 3). Membranes were incubated in secondary antibody for 1–2 h at room temperature (anti-mouse IgG (H + L) HRP conjugate (Bio-Rad) 1:1,000), developed (ECL Amersham) and imaged using ChemiDoc Touch Imaging System (Bio-Rad). All unedited blots are provided in Supplementary Fig. 10.