Iblot western blotting system

A total of 400 µg of each protein was resolved on a 4–12% gradient, 1.5 mm, 15-well Bis-Tris NuPAGE gel in 1X MES buffer (Invitrogen, ThermoFisher). SeeBlue Plus2 pre-stained protein standard (ThermoFisher) was included on the SDS-PAGE for molecular weight reference. Proteins were transferred onto nitrocellulose membranes using the iBlot Western blotting system (Invitrogen, ThermoFisher). Membranes were blocked overnight at 4 °C with 50 mL of 5% non-fat dry milk (Blotting-grade blocker, Bio-Rad Laboratories, Hercules, CA, USA) in 1X tris-buffered saline containing 0.1% Tween-20 (1X TBST). Membranes were probed with either anti-FLAG-alkaline phosphatase antibody (Sigma, A9469, St. Louis, MO, USA) or anti-polyhistidine-alkaline phosphatase antibody (Sigma-Aldrich, catalog #A5588) diluted 1:1000 in 5% non-fat dry milk in 1X TBST for 1 h at 4 °C. Membranes were washed 3 times for 5 min each at room temperature with 1X TBST. Membranes were developed using 1-step NBT/BCIP (ThermoFisher) according to the manufacturer’s recommendations.

Cells, EVs, and the supernatant of culture medium were lysed with IP lysis buffer (Thermo Fisher Scientific Inc., catalog number 87787) containing protease inhibitor (Roche Diagnostics, catalog number 04693159001). Protein concentration was measured using the bicinchoninic acid (BCA) method (Thermo Fisher Scientific Inc., catalog number 23225). Laemmli buffer (Boston Bioproduct; non-reducing, catalog number BP-110NR; reducing, catalog number BP-111R) was added to the lysate and boiled at 95 °C for 5 min. Total protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membrane using the iBlot Western blotting system (Life Technologies) or conventional wet method. Primary antibodies against human sortilin ICD (rabbit, 1:1000; Abcam plc, catalog number ab16640, lot number GR185198-1), human β-actin (mouse, 1:2000; Novus Biologicals, LLC, catalog number NB600-501, lot number 014M4759), FLAG (rabbit, 1:1000; Sigma, catalog number F4725, lot number 093M4798), His₆ (mouse, 1:1000; Abcam plc, catalog number ab18184, lot number GR247674-1) were used.

Macrophages whole cell lysate were prepared using RIPA buffer containing protease inhibitor (Roche). Total protein was separated by 4–20% Mini-PROTEAN TGX Precast Gel and transferred using the iBlot Western blotting system (Life Technologies). Primary antibodies against human GBP-1 (Abcam, Catalog# ab131255), WARS (Thermo Fisher Scientific, Catalog# PA5-29102), STAT1 (Cell signaling, Catalog# 9172), phosphorylated STAT1 at Y701 (Cell signaling, Catalog #9167,) JAK2 (Cell signaling, Catalog#3230), and β-actin (Novus) were used. Protein expression was detected using Pierce ECL Western Blotting substrate reagent (Thermo Scientific) and ImageQuant LAS 4000 (GE Healthcare).

Staphylococcal strains were grown in TSB at 37°C overnight. 10 µl of these cultures were inoculated into 1 ml of TSB and grown for 8 h. Culture supernatants were loaded onto 15% SDS-PAGE gels and run at 150 V for 1 h. Proteins in the gels were blotted on nitrocellulose membranes using an iBlot Western blotting system (Life Technologies, Grand Island, NY). Blotted membranes were incubated with Odyssey blocking buffer (LI-COR, Lincoln, NE) for 1 h at room temperature. Anti-staphylococcal α-toxin rabbit serum (1∶2,000, Sigma-Aldrich, St. Louis, MO) was added to the blocking buffer and incubated for another hour at room temperature. Membranes were washed five times with washing buffer (Tris-buffered saline containing 0.1% Tween-20, pH 7.4) and incubated with 1∶10,000 diluted Cy5-labeled goat anti-rabbit IgG (Life Technologies, Grand Island, NY) in Odyssey blocking buffer in dark for 1 h at room temperature. Membranes were washed five times with the washing buffer and scanned with a Typhoon TRIO+ Variable Mode Imager (GE Healthcare, Piscataway, NJ). The amount of α-toxin in the scanned image was quantified using ImageQuant TL software (GE Healthcare, Piscataway, NJ).

A human AV sample was pulverized in liquid nitrogen and resuspended in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, USA) with 1% protease inhibitor cocktail (Roche, Switzerland). The protein was precipitated and isolated in 6 M urea/2 M thiourea/100 mM TEAB (pH 8.0). Protein concentration was measured using the Pierce 660nm Protein Assay (Bradford) method (Thermo Fisher Scientific, USA). Human cortical protein (Takara Bio, Mountain View, CA, USA) and the AV protein were denatured and boiled in Laemmli buffer (Bio-Rad, Hercules, CA, USA). Total protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred using the iBlot Western blotting system (Life Technologies, Carlsbad, CA, USA). Primary antibodies against TUJ-1 (1:500; Abcam, USA) and GFAP (1:5,000; EMD Millipore, Billerica, MA, USA) were used. Protein expression was detected using Pierce ECL Western Blotting substrate reagent (Thermo Fisher Scientific, USA) and ImageQuant LAS 4000 (GE Healthcare, Arlington Heights, IL, USA).

MDA-MB-231 cells were pretreated with B6H12 (1 μg/ml) for 15 min. The cells were further treated with EGF for 7 min and total Lysate were performed using NP-40 lysis buffer as described above. CD47 and EGFR immunoprecipitation was performed using Dynabeads® Protein G Immunoprecipitation Kit (Life technologies) according to manufacturer's instructions with slight modifications in incubation time (3 h) for EGFR and CD47 antibodies with Dyna beads. The immunoprecipitated cell lysates were loaded on 4–12% NuPAGE gels (Life Technologies) and transferred using iBlot® - Western Blotting System (Life Technologies), The membrane was blocked with 3% milk with addition of Complete mini Pellet for 20 minutes. IP-western blots were performed using -EGFRY1068, EGFR and CD47 antibodies (1:1000) overnight at 4°C. The membrane was washed two times with TBST for 10 minutes. Secondary HRP (Amersham), IRDye 800 or 680 (LI-COR) 1:3000 were used for 1h at RT. The membranes were further washed 3 times for 10 minutes interval. The images were captured by using WesternSure PREMIUM Chemiluminescent Substrate with Odyssey® Fc(LI-COR). The membranes were immunoblotted with EGFR and CD47 for total IP-input.

Cells were lysed with RIPA buffer containing protease inhibitor (Roche). Protein concentration was measured using the bicinchoninic acid method (Thermo Scientific). Total protein was separated by 8–10% SDS–PAGE and transferred using the iBlot western blotting system (Life Technologies). Primary antibodies against human and mouse PARP14 (1:250, HPA01206 Sigma-Aldrich), human and mouse PARP9 (1:250, ab53796, Abcam), human and mouse STAT1 (1:1,000, #9172, Cell Signaling), phosphorylated STAT1 (1:1,000, #9167, Cell Signaling), human and mouse STAT6 (1:2,000, #9362, Cell Signaling), mouse (1:1,000, ab54461, Abcam) and human (1:2,000, #9361, Cell Signaling) phosphorylated STAT6 and human and mouse β-actin (1:5,000; Novus) were used. For secondary antibodies, we used anti-mouse (1:1,000–5,000, A4416, Sigma) and rabbit (1:1,000–5,000, NA934-1ML, GE Healthcare Life Sciences) IgG antibodies. Protein expression was detected using Pierce ECL Western Blotting substrate reagent (Thermo Scientific) and ImageQuant LAS 4000 (GE Healthcare). Uncropped images of western blots are demonstrated in Supplementary Figs 18–20.