Rab27a

Protoporphyrin IX (CAS: 553-12-8; purity, >95%) and tyrosinase derived from mushrooms (T128536) were purchased from Aladdin (Shanghai, China). Antibodies against tyrosinase (ab180753, 1:500), MITF (ab20663, 1:2,000), TRP-1 (ab3312, 1:1,000), TRP-2 (ab221144, 1:1,000), and cytokeratin (ab7753, 1:200) were purchased from Abcam (Cambridge, UK). p-CREB (9198S, 1:1,000) and the antibody against CREB (9197S, 1:1000) were obtained from Cell Signaling Technology (MA, USA). LY83583 (sc-200314A), KT5823 (sc-3534B), and antibodies against GP100 (sc-393094, 1:500), KIF5b (sc-133184, 1:500), myosin Va (sc-365986, 1:500), melanophinin (sc-365735, 1: 500), Rab27a (sc-74586, 1: 500), and Cdc42 (sc-8401, 1:500) were obtained from Santa Cruz Biotechnology (CA, USA). RT-qPCR kits were purchased from Takara Biomedical Technology (Beijing, China). The BCA protein assay kit (P0011), cell lysis buffer (P0013), cGMP assay kit, antibody against β-actin (AF0003), donkey anti-rabbit immunoglobulin G (IgG) (Alexa Fluor 555–labeled) (A0453, 1:500), and goat anti-mouse IgG (Alexa Fluor 488–labeled) (A0428, 1:500) were obtained from Beyotime Biotechnology (Shanghai, China).

Cells were detached by trypsinization and lysed in 0.2 mM HEPES (pH 7.4) containing 0.1% SDS, 1 mM Na₃VO₄ (phosphatase inhibitor) and protease inhibitor cocktail (10 µg/mL benzamidine, 2 µg/mL antipain, 1 µg/mL leupeptin and 1 mM PMSF). Purified exosomes were sonicated in 0.9% NaCl containing 1 mM PMSF. Protein concentration was quantified with the Bradford microplate system Gen5TM EPOCH (BioTek). Total protein extracts (30 µg/lane) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF). Membranes were blocked with 5% skim milk in PBS/0.1% Tween (PBST) and then probed with antibodies against CD9 (m-monoclonal; Santa Cruz; 1:200), TSG101 (m-monoclonal; Santa Cruz; 1:200), ALIX (m-monoclonal; Cell Signaling; 1:500), Rab27a (m-monoclonal; Santa Cruz; 1:100), Calnexin (r-polyclonal; Abcam; 1:1000), HSP60 (r-polyclonal; Abcam; 1:20000), VE-Cadherin (m-monoclonal; Abcam; 1:1000), Lactadherin (m-monoclonal; Santa Cruz; 1:1000) and HSP90 (m-monoclonal; Abcam; 1:1000). Bound antibodies were then detected with peroxidase-labeled anti-mouse (Calbiochem 1:5000) or anti-rabbit (Calbiochem 1:3000) IgG. Blots were revealed with the EZ-ECL system (Biological Industries) on a C-DiGit Blot Scanner (LI-COR Biosciences)^{34 (link),36 (link)}.

Melan-a melanocytes were lysed with RIPA buffer and 1X protease inhibitor cocktail (PIC) (Sigma-Aldrich). The protein extracts were separated at 200 V using SDS-PAGE and the separated proteins were then transferred to PVDF membranes (Pall, Glen Cove, NY, USA) at 25 V for 2 h. To analyze protein expression, the PVDF membranes were immunoblotted overnight at 4 °C with antibodies to β–actin (Sigma), MyoVa (Cell Signaling Technology, Danvers, MA, USA), Rab27a (Santa Cruz, Dallas, TX, USA), Mlph (Protein Tech Group Inc., Chicago, IL, USA), Tubulin (Cell Signaling Technology) and GR (Cell Signaling Technology). Secondary horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Bethyl Laboratories, Montgomery, TX, USA) or anti-mouse antibodies (Bio-Rad, Hercules, CA, USA) were then incubated at room temperature for 1 h. Protein expression was detected using chemiluminescence ECL solution (Thermo, Waltham, MA, USA) and visualized with Chemi-Doc XRS (Bio-Rad).