Rab27a

Cell lysates or mice skin homogenates were extracted using lysis buffer (10 mM Tris–HCl, 1 mM ethylene diamine tetra acetic acid (EDTA), 1% sodium dodecyl sulfate, 1% Nonidet P-40, 1 : 100 proteinase inhibitor cocktail, and 50 mM β-glycerophosphate, and 50 mM sodium fluoride) (Beyotime, Shanghai, China). The protein concentration was determined with a protein assay kit (Beyotime, Shanghai, China), following the manufacturer's instructions. Aliquots of 40 to 50 μg per sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocked with 5% bovine serum albumin (BSA) in PBST (PBS with 0.1% Tween). Then, they were incubated with the following primary antibodies overnight: RELM-α (Santa Cruz Biotechnology, Dallas, Texas, USA), CD63 (Santa Cruz Biotechnology, Dallas, Texas, USA), CD81 (Santa Cruz Biotechnology, Dallas, Texas, USA), Rab27a (Abcam, Cambridge, UK), pknox1 (Abcam, Cambridge, UK), and anti-GAPDH (Abcam, Cambridge, UK). Then, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China). The blots were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA), according to the manufacturer's instructions.

Proteins derived from cells and EVs were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto nitrocellulose membranes, bound with primary antibodies, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence detection reagents (34580, Thermo Scientific, Waltham, MA, USA). Primary antibodies against the following proteins were used: Alix (ab56932, Abcam, Cambridge, UK), CD63 (ab68418, Abcam), TSG101 (ab56932, Abcam), Synthenin-1 (ab133267, Abcam), β-actin (112620, Cell Signaling Technology, Danvers, MA, USA), PD-L1 (13684, Cell Signaling Technology), Rab27a (ab55667, Abcam), Rab11 (ab18211, Abcam), Rab7 (ab50533, Abcam), pan Ras (sc-166691, Santa Cruz, Santa Cruz, CA, USA), Rap1A (sc-398755, Santa Cruz), p-B-Raf (2696S, Cell Signaling Technology), B-Raf (9433S, Cell Signaling Technology), p-MEK (9154S, Cell Signaling Technology), MEK (9126S, Cell Signaling Technology), p-ERK1/2 (4370S, Cell Signaling Technology), ERK1/2 (4695S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), and AKT (9272S, Cell Signaling Technology).

Western blotting was performed as described previously²² . In brief, whole-cell lysates or exosomal proteins were separated by SDS–PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% milk for 1 h at room temperature and then incubated with the corresponding primary antibodies overnight at 4 °C. The following antibodies were used: TSG101 (sc-136111, Santa Cruz, CA, USA), CD81 (sc-23962, Santa Cruz), Alix (92880, CST, MA, USA), ERK (4695, CST), p-ERK (4370, CST), Akt (9272, CST), p-Akt (4051, CST), STAT3 (12640, CST), p-STAT3 (9145, CST), EphA2 (6997, CST), EphA2 (398832, Santa Cruz), Rab27a (ab55667, Abcam, MA, USA), and β-actin (A1978, Sigma-Aldrich, MO, USA). After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The signals were visualized with the ECL kit. CD81, Alix, and TSG101 were used as exosomal markers. β-actin was used as a loading control.

The following antibodies were used against perforin (dG9), lysosome-associated membrane protein (LAMP) 1–Alexa Fluor 488 (H4A3), CD3 (UCHT1), CD56 (HCD56), and NKG2D (1D11; BioLegend, San Diego, Calif); cation-independent mannose 6-phosphate receptor (CI-MPR; 2G11), dynein heavy chain, and Ras-associated binding protein (Rab) 27a (Abcam, Cambridge, United Kingdom); early endosome antigen 1 ([EEA-1] 14/EEA1; BD Biosciences, San Jose, Calif); cathepsin D, Rab7, and Rab9 (Cell Signaling, Danvers, Mass); LAMP1 (H4A3), LAMP2 (H4B4), granzyme B (GB7), Munc13-4, and Rab14 (D-5; Santa Cruz Biotechnology, Dallas, Tex); myosin IIA and actin (AC-15; Sigma, St Louis, Mo); vesicle associated membrane protein 7 (Synaptic Systems, Goettingen, Germany); LAMP2–Alexa Fluor 488 (H4B4) (eBioscience, San Diego, Calif); and perforin (Pf-344, for immunoblotting; Mabtech, Stockholm, Sweden).

Cellular or EV proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with the respective primary antibody against CD63 (1:1000; Abcam), beta-actin (1:20000; CST), ALIX (1:1000; CST), flotillin-1 (1:1000, CST), RAB27A (1:1000; Abcam), ETA (1:2000; Abcam), Akt (1:2000; CST), phospho-Akt (1:2000; CST), or PD-L1 (1:1000; CST). After removing the primary antibody and three washing steps at 10-min intervals, the blots were incubated with a horseradish peroxidase (HRP)-linked secondary antibody. The images were visualized using enhanced chemiluminescence (ECL) detection reagents (Thermo Scientific) and quantified using ECL hyper-film (AGFA) and Fusion FX7 system (Vilber Lourmat).