Gtx109669

Whole cell protein lysates were prepared with RIPA buffer – 25 mM Tris HCl, 150 mM NaCl, 1% v/v NP-40, 1% w/v sodium deoxycholate, and 0.1% w/v sodium dodecyl sulfate; pH 7.4) with 1× Halt™ protease/phosphatase inhibitor cocktail (Thermo Scientific^®). Protein lysates (10 μg per lane) were subjected to standard denaturing, reducing polyacrylamide gel (10%) electrophoresis and transferred to a polyvinyl-difluoride membrane. Tris-buffered saline (20 mM Tris HCl and 150 mM NaCl; pH 7.4) with 0.05% v/v Tween-20 and 5% w/v non-fat dry milk was used as buffer to probe the membrane with antibodies against BIRC5 (71G4B7 rabbit monoclonal antibody, product 2808, Cell Signaling Technology^®, Danvers, MA; used at 1:1000 dilution) or calnexin (rabbit polyclonal antibody, product GTX109669, GeneTex^®, Irvine, CA; used at 1:5000 dilution). Antibodies bound to their targets on the membrane were detected through chemiluminescence and its detection on radiographic films following the binding of a horseradish peroxidase-conjugated goat antibody against anti-rabbit IgG (product 31460, Thermo Scientific^®). Intensities of protein bands were determined by densitometry of scanned radiograms with NIH ImageJ software.

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.

FLAG-tagged Tg in pcDNA3.1+/C-(K)-DYK plasmid was purchased from Genscript (Clone ID OHu20241). Site-directed mutagenesis was then performed to generate FT-G2341R, FT-L2284P, FT-C1264R, FT-A2234D, and untagged Tg plasmids (supplemental Table S1). Primary antibodies were acquired from commercial sources and used at the indicated dilutions in immunoblotting buffer (5% bovine serum albumin in Tris-buffered saline pH 7.5, 0.1% Tween-20, and 0.1% sodium azide). Mouse monoclonal antibodies were used for the detection of KDEL (1:1000, Enzo Life Sciences, ADI-SPA-827), M2 anti-FLAG (1:1000, Sigma Aldrich, F1804). Polyclonal rabbit antibodies were used to detect Calnexin (1:1000, GeneTex, GTX109669), protein disulfide isomerase family A member 4 (PDIA4) (1:1000, Proteintech, 14712-1-AP), DNAJC10 (1:500, Proteintech, 13101-1-AP), thyroglobulin (1:1000, Proteintech, 21714-1-AP), UGGT1 (1:1000, Proteintech, 14170-1-AP), STT3A (1:2000, Proteintech, 12034-1-AP), and STT3B (1:2000, Proteintech, 15323-1-AP). Secondary antibodies were obtained from commercial sources and used at the indicated dilutions in 5% milk in Tris-buffered saline pH 7.5, 0.1% Tween-20 (TBS-T): Goat anti-mouse Starbright700 (1:10,000, Bio-Rad,12004158), Goat anti-rabbit IRDye800 (1:10,000, LI-COR, 926-32211), and Goat anti-rabbit Starbright520 (1:10,000, Bio-Rad,12005869).