Rab27a

Briefly, extracted proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% milk incubated with the primary antibodies against the following: Alix (Bethyl Laboratories, A302-938A), Calretinin (Swant, CG1), CD63 (Santa Cruz Biotechnology, SC-15363), Flotillin1 (Abcam, AB41927), Porin (Calbiochem, 529536), Rab27a, TSG101 (Genetex, GTX70255), β-Actin (Sigma–Aldrich, A5441) overnight at 4°C, and finally incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology). Protein bands were visualized using SuperSignal^® West Pico Chemiluminescent Substrate (Millipore) and imaged using a Fuji-film LAS-3000/4000. Band densities were analyzed using ImageJ software. Protein levels were corrected to β-Actin. Protein data were analyzed and presented as for PCR data (see above).

The cells were lysed at 4 °C in RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts (30 μg) of protein were separated electrophoretically using SDS-polyacrylamide gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). After being blocked with 5% non-fat milk, the membrane was incubated with specific antibodies (RAB3C: GTX108610, 1:5000, GeneTex, Taipei, Taiwan; RAB27A: GTX109180, 1:5000, GeneTex, Taipei, Taiwan; RAB3B: GTX108610, 1:5000; GeneTex, Taipei, Taiwan; RAB26: GTX118872, 1:5000; GeneTex, Taipei, Taiwan; IL-6: GTX110527, 1:1000, GeneTex, Taipei, Taiwan; STAT3: #4904, 1:1000, Cell Signaling, USA; phospho-STAT3: #9145, 1:1000, Cell Signaling, USA) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The blots were visualized using an ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).