Nupage bis tris pre cast gels

Cells were collected in Pierce RIPA buffer (Thermo Fisher Scientific) plus HALT protease inhibitor cocktail (1:100) (Thermo Fisher Scientific) and lysates loaded on 12% NuPage Bis-Tris pre-cast gels (Thermo Fisher Scientific). After separation by electrophoresis, proteins were transferred to 0.2 μm nitrocellulose membranes (Thermo Fisher Scientific). Membranes were blocked with 5% milk in TBS +0.1% Tween and incubated with primary antibody overnight. Information for primary antibodies is provided in Table S2. Membranes were washed and incubated with secondary antibody for 1 h at room temperature in 5% milk-TBS-0.1% Tween and developed using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific). Human β-Actin was employed as an internal reference.

MV and EXO lysates were prepared in RIPA buffer with Halt^TM Protease and Phosphatase inhibitor cocktail, EDTA-free 100× (Thermo Fisher). MV and EXO lysates were loaded in 4–12% NuPAGE Bis–Tris precast gels (Thermo Fisher), proteins were separated and transferred onto a nitrocellulose membrane (Amersham Protran, USA). Membranes were incubated overnight at 4 °C with the following antibodies: anti-P2X7 (1:300, P8232, Sigma-Aldrich), anti-calnexin (1:5000, GTX109669, GeneTex), anti-Alix (3A9) and anti-flotillin-1 (1:1000, Exosomal Marker Antibody Sampler Kit, Cell Signaling Technology). Secondary anti-rabbit (1706515, BIORAD) or anti-mouse (1706516, BIORAD) antibodies were applied at a 1:3000 dilution.

Flash frozen tumors were pulverized to powder by mechanical force (Covaris cryoPREP). Tumor powder was lysed with RIPA lysis buffer (Sigma) supplemented with a cocktail of phosphatase and protease inhibitors (Thermo Fisher Scientific) and 1 mM PMSF (Sigma). Tumor lysates were homogenized by mechanical disruption (SPEX SamplePrep) and cleared by centrifugation at 14,000 rpm for 10 min at 4 °C. Protein levels were quantified by the BCA Protein Assay Kit (Pierce Biotechnology) and normalized to equal concentrations. Equal amounts of protein lysates were resolved using NuPAGE Bis-Tris precast gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes using the iBlot® dry blotting system (Thermo Fisher Scientific). Membranes were blocked with blocking buffer for fluorescent Western blotting (LI-COR). Primary antibodies were detected using IR Dye 800-conjugated (LI-COR) or IR Dye 680-conjuated (LI-COR) species-selective secondary antibodies. Detection and quantification were conducted using an Odyssey infrared scanner (LI-COR) using the manufacturer’s software. Protein loading was assessed using antibodies to β-actin or GAPDH.

Cell lysates or tumor homogenates were loaded in 4–12% NuPAGE Bis-Tris precast gels (Thermo Scientific) and proteins were separated and transferred onto a nitrocellulose blotting membrane (Amersham Protran, GE Healthcare, USA). Membranes were incubated overnight at 4 °C with primary antibodies as follows: anti-P2X7 (P8232, Sigma-Aldrich) antibody, recognizing the C terminal domain of the protein was diluted 1:300, anti-P2X7 (APR008, Alomone Labs, Jerusalem, Israel) antibody, recognizing the extracellular loop of the protein was diluted 1:300, anti-c-myc antibody (A190-105A, Bethyl Laboratories, INC) was diluted 1:2500, and anti-SKP-1 antibody (MA5-15928, Thermo Scientific) was diluted 1:1000. Membranes were then incubated with secondary goat anti-rabbit (170-6515, BIORAD) or goat anti-mouse (170-6516, BIORAD), HRP-conjugated antibodies at a 1:3000 dilution. When required blocking peptide for the APR008 antibody (BLP-PR008, Alomone Labs) was added to the primary antibody at a 1:2 ratio and preincubated for 5 h before proceeding with the above-described immunostaining protocol. Protein bands were visualized by ECL HRP Chemiluminescent Substrate ETA C ULTRA 2.0 (Cyanagen Srl, Bologna, Italy) with a Licor C-Digit Model 3600. Densitometric analysis was carried out with ImageJ software and data were normalized on SKP-1 content.

Protein extracts were prepared by scraping the cells in the lysis buffer containing 100 mM KCl, 5 mM MgCl2, 20 mM Hepes-KOH (pH 7.8), 2 mM DTT, 25% NP-40, and a protease inhibitor cocktail (Roche). 20 μg of the whole protein extract was loaded onto 4–12% NuPAGE Bis-Tris Pre-Cast Gels and resolved using the NuPAGE System followed by iBlot transfer (ThermoFisher). Primary antibodies were: CPEB1 dilution 1:1000 (ab73287 Abcam) TWIST1 H-81, dilution 1:1000 (sc-15393, Santa Cruz Biotechnology), anti-c-Myc 9E10, dilution 1:1000 (11 667 149 001 ROCHE), anti-ZEB1 OTI3G6, dilution 1:1000 (TA802298 Origene), β-Actin (C4) HRP, dilution 1:2000 (sc-4778 HRP Santa Cruz Biotechnology).