Anti src ab 1

Cells and exosomes were lysed in n-octyl-β-d-glucoside buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 20 mM NaF, 1% Nonidet P-40, 5% glycerol, 2% n-octyl-β-d-glucoside, and protease inhibitor cocktail), and immunoblotting was performed as described previously^{40 (link)}. The following antibodies were used: anti-Alix (ABC40, Merck, Darmstadt, Germany), anti-TSG101 (C-2, Santa Cruz, Santa Cruz, CA, USA), anti-CD63 (MX-49.129.5, Santa Cruz), anti-phospho-tyrosine (pY,4G10, Merck), anti-Src pY416 (D49G4, Cell Signaling Technology, Danvers, MA, USA), anti-Src (Ab-1, Merck), and anti-GAPDH (6C5, Santa Cruz). Anti-Nluc rabbit polyclonal antibody was kindly provided by Promega (Madison, WI, USA).

Cells were lysed with a 1% Triton X-containing lysis buffer, as described previously [22 (link)]. Lysates were boiled with sodium dodecyl sulfate (SDS) sample buffer for 5 min at 95 °C [22 (link)]. Samples were separated on 7.5, 10, 12 and 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies. The antibodies used were anti-Plectin-1 (#12254), anti-Src pY416 (#2101), anti-Pyk2 (#3292), anti-Pyk2 pY402 (#3291), anti-p38 MAPK (#8690), anti-phospho-p38 MAPK (Thr180/Tyr182) (#9211), anti-p44/42 MAPK (Erk1/2) Antibody (#9102), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370) (Cell Signaling Technology, CST, Danvers, MA), anti-Src (ab-1) (Merck, Darmstadt, Germany), anti-DDDDK-tag (Flag-tag, Fla-1), anti-GAPDH mAb-horseradish peroxidase-conjugated (HRP)-DirecT (Medical & Biological Laboratories, MBL, Tokyo, Japan), and β-actin (A2228, Sigma Aldrich Chemicals, St. Louis, MO). The membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies. Finally, the blots were imaged using a LAS4000 (Fujifilm Wako) with Immobilon ECL Ultra Western HRP Substrate (Merck).