Anti cd63

Microvesicle and exosome fractions from 16 ml urine samples were separated by SDS-PAGE (8–16% acrylamide gradient) and then transferred to a nitrocellulose membrane. The membrane was blocked with 1% bovine serum albumin (BSA) in PBS plus 0.1% Tween-20 (PBST), rinsed in PBST, and labeled with one of the following primary human antibodies diluted in 1% BSA in PBST: anti-CD63 (Novus Biological, Littleton, CA, USA), anti-CD81 (Novus Biological, Littleton, CA, USA), or anti-CD45 (LifeSpan BioSciences, Seattle, WA, USA) [41 (link)]. After rinsing again in PBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (Novus Biological, Littleton, CA, USA). Chemiluminescence was detected and quantified using the ChemiDoc Touch Imaging System and Image Lab software (Bio-Rad, Hercules, CA, USA).

Expression and subcellular localization of EGFR, phosphorylated ERK (p42/44) and, phosphorylated p38 was tested by indirect immunofluorescence staining and confocal microscopy. Staining was done on FFPE tissue sections. The antibodies used were anti-EGFR (Cell Signaling, #42675), anti-phospho-ERK (Cell Signaling, RRID-AB-331775), anti-p38 (Cell Signaling, RRID-AB2139682), anti-ALIX (NOVUS, RRID-AB-960843) and, anti-CD63 (NOVUS, RRID-AB-108402). Primary antibodies were diluted according to manufacturers’ recommendations and incubated at 4°C overnight. Secondary antibodies labeled with Alexa Fluor 555 and Alexa Fluor 488 (Invitrogen), were incubated for one hour at room temperature. The nonspecific background was removed using blocking solution (DAKO). DAPI was used to counter stain the nuclei. Immuno-stained sections were visualized by laser scanning confocal microscope (LSM 700, Zeiss, Germany). Colocalization coefficients were calculated by Zen software (Zeiss, version 14.0.0.201, Germany) based on the co-occurrence of the red and green signals and their relative intensities. Colocalization coefficient of >0.5 was considered as positive co-expression.

Western blotting was performed as described previously (Hosoi et al., 2014 (link)) albeit with some modifications. Briefly, the cells were washed and lysed for 20 min with RIPA buffer containing 25 mM Tris HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate, 1 mM Na₃VO₄, 10 mM NaF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were incubated on ice for 20 min. Then, the samples were sonicated for 4 min and suspended for 30 min at 4°C. After additional sonication for 4 min, the samples were centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were collected. The samples were then boiled in SDS sample buffer for 3 min, fractionated with SDS-PAGE, and transferred to nitrocellulose membranes. These membranes were then incubated with anti-CD63 (Novus; 1:1000), anti-calnexin (Enzo; 1:1000), anti-CD81 (Cell signaling; 1:1000), and anti-IRE1α (Cell signaling; 1:1000) antibodies, followed by an anti-horseradish peroxidase-linked antibody. Peroxidase binding was detected using chemiluminescence with an enhanced chemiluminescence system.

CD63 expression was quantitatively analyzed using Western blotting. Purified exosomes were mixed with 5% sodium dodecyl sulfate (SDS) sample buffer (Tech & Innovation, Gangwon, Republic of Korea), and sliced tissues were lysed and homogenized in radio-immunoprecipitation assay buffer (Tech & Innovation, Republic of Korea) containing protease inhibitors. The protein concentrations of the separated solutions were measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). The samples were heated for 10 min at 97 °C. Next, 15 µg of protein from each sample was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) for 90 min. Each membrane was blocked with 5% skim milk (BD Life Sciences, Franklin Lakes, NJ, USA) in TBST buffer (25 mM Tris, 190 mM NaCl, and 0.05% Tween 20, pH 7.5) for 1 h at room temperature, followed by incubation with primary antibodies (anti-CD63, 1:300; Novus Biologicals, Centennial, CO, USA) at 4 °C overnight. After five 20 min washes with TBST, each membrane was washed three times in Tween-20 and incubated with goat anti-mouse IgG for 2 h. Bands were visualized using an enhanced chemiluminescence system; the intensity of the blots was quantified with Image J 1.44 software (https://imagej.nih.gov/ij/plugins/index.html) [43 (link)].