Anti cd63 antibody

Baby green monkey kidney (BGMK) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). BGMK cells were cultured in Dulbecco-modified Eagle’s medium (DMEM; HyClone, Ottawa, ON) with 7 % fetal bovine serum (FBS; HyClone), 2-mM L-glutamine, 100-U/mL penicillin, and 100-μg/mL streptomycin, at 37 °C with 5 % CO2. The following antibodies/probes were used during immunofluorescence: Lysotracker®DND-99 from Molecular Probes (Burlington, ON), the 9E10 mouse and mouse myc monoclonal antibody from Roche (dilution 1/100; Indianapolis, IN), rabbit anti-FLAG (dilution 1/100; Burlington, ON), and FITC/Cy3-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (IgG) from Jackson ImmunoResearch Inc. (dilutions 1/100, 1/200 respectively; West Grove, PA, anti-CD63 antibody from Invitrogen (dilution 1/100; Burlington, ON); and MitoTRACKER Red FM from Molecular Probes (Burlington, ON). LITAF WT was synthesized by GenScript (Piscataway, NJ). Myc-tag was added to the N-terminus of the protein to facilitate imaging. PMP22 was synthesized by Sino Biological Inc. (BC019040, Beijing). The gene was pCMV/hygro and contained a flag-tag.

Protein quantification was performed using Pierce BCA Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s recommended microplate assay procedure. Absorbance was measured with a Spectra Max M5 multimode microplate reader using Soft Max Pro data acquisition and analysis software (Molecular Devices, Sunnyvale, CA, USA). Vesicle isolates were denatured in 4X Laemmli sample buffer at 95°C for 10 min. Proteins were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in Tris/Glycine/SDS running buffer and transferred to Immun-Blot PVDF membrane (all reagents and supplies from Bio-Rad, Hercules, CA, USA). Immunoblotting was performed with the anti-CD63 antibody (Invitrogen by Life technologies, Carlsbad, CA, USA). Primary antibody incubation time was 16 h at 4°C (overnight). Washing was done with PBST for three times, and further blots were washed with PBS and incubated with appropriate HRP-conjugated secondary antibody for 2 h at 4°C (A9917, Sigma-Aldrich, MO, USA). The 3, 3’ Diaminobenzidine (DAB) was used for developing the membrane. Densitometry analysis was performed on digitized images using Image J image processing and analysis software.