Anti rab27a

For western blotting, immunoprecipitation and immunofluorescence, the following antibodies were used: anti-ANXA2 (Abcam, ab54771, for confocal microscopy; BD, 610069, for western blotting), anti-CD63 (Abcam, ab18235), anti-Tsg101 (Abcam, ab83), mouse IgG (BD, 554121), anti-ATG5 (Cell Signaling Technology, 2630), anti-calnexin (Cell Signaling Technology, 2433), anti-LAMP1 (Cell Signaling Technology, 9091), anti-RAB7 (Cell Signaling Technology, 9367), anti-LC3 (MBL, PM036), anti-RAB8A (Proteintech, 55296-1-AP), anti-RAB27A (Proteintech, 17817-1-AP), anti-RAB27B (Proteintech, 13412-1-AP), anti-VAMP7 (R&D Systems, MAB6117), anti-cathepsin S (Santa Cruz, sc-74429), anti-SQSTM1/p62 (Santa Cruz, sc-28359), and anti-α-tubulin (Santa Cruz, sc-5286). For secondary staining in western blotting, the following antibodies were used: HRP-linked anti-mouse IgG (Cell Signaling Technology, 7076) and HRP-linked anti-rabbit IgG (Cell Signaling Technology, 7074). For secondary staining in immunofluorescence, the following antibodies were used: Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, A11034), Alexa Fluor 594 donkey anti-mouse IgG (Life Technologies, A21203), Alexa Fluor 594 donkey anti-rabbit IgG (Life Technologies, A21207), Alexa Fluor 647 donkey anti-mouse IgG (Life Technologies, A31571), and Alexa Fluor 647 donkey anti-rabbit IgG (Life Technologies, A31573).

Cells were lysed in 100ul RIPA buffer (Beyotime, Shanghai, China) with 1% PMSF (Beyotime, Shanghai, China). Protein sample was separated by 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% milk in Tris-buffered saline for 1h at room temperature, followed by incubation with primary antibodies at 4°C overnight, and subsequent incubation with secondary antibodies at room temperature for 1h. The antibodies used for this study included anti-ERK1/2 (proteintech, 1:800), anti-phosphorylation ERK (proteintech, 1:3000), anti-RAB27A (proteintech, 1:2000), anti-β-actin (proteintech, 1:2000) and goat anti-rabbit IgG (proteintech, 1:2000). Results were analyzed by ImageJ version 6.0. Three independent experiments were done for statistical analysis.

One million PBMCs per donor were lysed in RIPA buffer supplemented with 1 × protease inhibitor cocktail (Santa Cruz Biotechnology) for 30 min on ice. Supernatants were mixed with 4 × NuPage loading buffer (Invitrogen) added 10 mM DTT (Invitrogen), run on a 4–12% Bis–Tris gel (Invitrogen), and transferred to a nitrocellulose membrane (iBlot, Invitrogen). Rabbit polyclonal anti-RAB27A (Proteintech Group) and HRP-conjugated goat anti-rabbit secondary antibodies (Invitrogen) were used for detection. A directly HRP-conjugated mouse anti-actin antibody (Sigma) was used as loading control. Blocking buffer and antibodies were diluted in 5% non-fat dry milk (Biorad) in TBS-Tween 0.2%.