Irdye 800cw goat anti mouse

Cell lysates were harvested with RIPA lysis buffer supplemented with 1 mM NaF, complete EDTA-Free Protease Inhibitor Cocktail (Sigma-Aldrich), and 2 mM Na₃VO₄. Proteins were resolved in 10% SDS-PAGE gels and transferred onto a 0.45 µm nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies at 4 °C overnight, followed by secondary antibodies LI-COR IRDye 680RD goat anti-Rabbit (#926- 68071, 1:10,000 dilution) or LI-COR IRDye 800CW goat anti-Mouse (#926-32210, 1:10,000 dilution) and scanned on LI-COR Odyssey CLx (Lincoln, NE). At least two independent immunoblots were performed for each experiment, with a representative immunoblot shown. Antibodies are listed in Supplementary Table 2.

Ten µL aliquots of either a UC preparation or individual SEC fractions were analyzed by Western blot against human CD71. Membranes were probed with rabbit polyclonal anti-CD71 (ab84036) at 1:250 dilution for 1 hour at RT. A goat anti-rabbit IgG coupled to HRP (Sigma, A6154) was used at a dilution of 1:2,500 for 1 hour at RT. Revealing was performed using ECL Western Blotting Substrate (Pierce™) in ImageQuant LAS 4000 (GE Healthcare Life Sciences). Additionally, 20 µL aliquots of UC preparations were analyzed to confirm the presence of HSP70, GAPDH and stomatin in HuRex. Membranes were incubated for 1 hour at RT with primary antibodies anti-HSP70 (Santa Cruz Biotechnology, W27 sc-24) at 1:250 dilution, anti-GAPDH (Sigma, G9545) at 1:500 dilution or anti-stomatin (Invitrogen, PA5-30019) at 1:250 dilution. Subsequently, membranes were washed and incubated for 1 h at RT with the Li-Cor IRDye-labeled secondary antibodies IRDye® 800CW goat-anti-mouse (925-32210, Li-Cor Biosciences) at 1:15,000 dilution or IRDye® 680RD goat anti-rabbit (925-68021, Li-Cor Biosciences) at 1:20,000 dilution. Blots were analyzed with the Odyssey near-infrared system (Li-Cor Biosciences) having the intensity of 700 channel set up at 5 and the one of 800 channel at 7. Images were edited using the software Image J (NIH).

Immunoblots was performed following the Licor-system western blot detection protocol (licor.com/bio/support). 20ug of whole cell lysate from LNCaP and HL60 cells were ran in 4–20% SDS-page gel (Biorad, Cat# 4568094) and transferred to Immobilon-FL Transfer Membrane PVDF 0.45 uM pore size (Immobilon cat# IPFL00010). Protein was probed with antibodies against B7-H3 (Invitrogen, Cat # MAS-15693; R&D Systems, Cat # AF1027; Bethyl Laboratories, Cat # A700-026) and GAPDH (Santa Cruz, Cat # sc-32233). Precision plus was used as the protein standard (Bio-Rad, Cat# 161-0363). Primary antibodies were diluted 1:500 and incubated in 5% BSA, PBX and 0.2% tween-20 blocking solution. Secondary antibodies (IRDye 800 CW Goat-anti Mouse, Licor, Cat # 925-32210; IRDye 800 CW Goat-anti Rabbit, Licor, Cat # 925-32211; HRP-Conjugated Mouse-anti Goat, Santa Cruz, Cat # SC2354) were diluted 1:2000 and incubated in 5% BSA, 1× PBX, and 0.2% tween-20 and 0.01% SDS. Bio-rad Chemi-Doc MP Imaging system was used to scan the PVDF membrane for HRP activity detection. The protein expression of B7-H3 was normalized to GAPDH (loading control). All blots were derived from the same experiment and they were processed in parallel. The original scans of the blots are shown in Supplemental Fig. 4.

Cells were lysed with RIPA Buffer. Proteins were separated on a 10% Criterion TGX Gel (BioRad) and transferred to PVDF membrane before blocking with 5% Skim Milk in TBST (Tris-buffered Saline, 0.1% Tween-20). Primary antibodies used were against GATA3 (Abcam, ab199428) and alpha-Tubulin (Sigma, B512), secondary antibodies used were goat anti-rabbit HRP (Cayman Chemical) and IRDye 800CW goat anti-mouse (LI-COR, 926-32210). Chemiluminescence was imaged using an Amersham Imager 600 (GE), and fluorescence was imaged using an Odyssey CLx (LI-COR).

Cells and sEV were lysed with Pierce™ RIPA lysis buffer (Pierce Protein Biology/Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor (Sigma‐Aldrich) and protein content determined using the BCA protein assay (Pierce Protein Biology/Thermo Fisher Scientific). Polyacrylamide gel electrophoresis (PAGE) was performed using standard reagents from Invitrogen/Thermo Fisher Scientific. Samples were denatured for 5 min at 95°C, loaded onto Bolt™ 4–12% Bis‐Tris Plus Gels, run at 200 V for 28 min and transferred to a nitrocellulose membrane at 10 V for 60 min. Membranes were blocked for 1 h at room temperature using Intercept^® (TBS) blocking buffer (LI‐COR, Lincoln, NE, USA) and subsequently probed with primary antibodies overnight at 4°C, including CD63 (Mouse monoclonal IgG1, Clone Ts63; Invitrogen/Thermo Fisher Scientific), CD9 (Mouse monoclonal IgG1, Clone Ts9; Invitrogen/Thermo Fisher Scientific), CD81 (Mouse IgG1, Clone M38; Invitrogen/Thermo Fisher Scientific) and Calnexin (Mouse monoclonal IgG1, Clone AF18; Santa Cruz, Dallas, TX, USA). Proteins were visualized with IRDye 800CW goat anti‐mouse (LI‐COR) using the Odyssey CLX (LI‐COR). Quantitative analysis of protein intensities was performed using Image Studio 5.2 software (LI‐COR).

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on Mini-Protean TGX Precast protein gels (Bio-Rad, Hercules, CA, USA) and transferred onto nitrocellulose membranes (Bio-Rad) using a Turbo transfer system (Bio-Rad). Membranes were blocked with 5% BSA T-BST (500 mM Tris HCl [pH 7.5], 1.5 M NaCl, 0.05% Tween 20). SpCas9-HF1 and β-actin were detected with antibodies 7A9-3A3 (Cell Signaling Technology, Danvers, MA, USA) and GTX629630-25 (GeneTex, Irvine, CA, USA), respectively. Membranes were probed with the fluorescent secondary antibodies IRDye 800CW goat anti-mouse (925-32210, LI-COR Biosciences, Lincoln, NE, USA) and IRDye 680RD goat anti-rabbit (925-68071, LI-COR Biosciences), respectively, and bands were visualized with a ChemiDoc MP Imaging System (Bio-Rad).