Caveolin 1

The human neuroblastoma cell line SK-N-MC was obtained from Korean Cell Line Bank (Seoul, Korea). The antibodies of p-Akt (Thr308), p-Akt (Ser473), Akt, mTOR, Caveolin-1, Flotillin-2, p-Tau (Ser396), Tau, p-NF-κB p65 (Ser536), NF-κB p65, Lamin A/C, CBP and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies of p-mTOR (Ser2448), p-p70S6K1 (Thr389) and p70S6K1, were acquired from Cell Signaling Technology (Beverly, MA, USA). Aβ, BACE1, HIF-1α and GPR40 antibodies were obtained from Abcam (Cambridge, MA, USA). The C99 antibody was purchased from EMD Millipore (Darmstadt, Germany). Horse radish peroxidase (HRP)-conjugated IgG was obtained from Jackson Immunoresearch (West Groove, PA, USA). PA, BSA, GW9508, ionomycin, PF4708671, LY294002 and rapamycin were purchased from Sigma Chemical Company (St. Louis, MO, USA). The Akt inhibitor, GW1100 and SN50 used here were purchased from Calbiochem (La Jolla, CA, USA). siRNAs for GPR40, GPR120, APP, BACE1, HIF-1α and non-targeting were obtained from Dharmacon (Lafayette, CO, USA).

Following exposure to shear, cells were washed three times with ice-cold phosphate-buffered saline (PBS) and lysed with radioimmunoprecipitation assay buffer (RIPA) as described previously [35 (link)]. The lysate was further homogenized by sonication. The protein content of each sample was determined by Bio-Rad DC assays. Aliquots of cell lysate (20 μg of protein) were then resolved by size on 10% SDS-polyacrylamide gels and subsequently transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with a primary antibody overnight at 4°C, followed by incubation with an alkaline phosphatase-conjugated secondary antibody for 1 h at room temperature. Protein expression was detected using chemiluminescence, and the intensities of immunoreactive bands were determined via densitometry and the NIH ImageJ program [36 (link)]. Primary antibodies specific for KLF2, eNOS (BD Biosciences), β actin (Santa Cruz), VCAM-1 (Santa Cruz), Flk-1 (VEGF-R2, Santa Cruz), and Caveolin-1 (Santa Cruz) were used.

Specific siRNA targeting TRIF (sc-154266), TRAM (sc-44748), TIRAP (sc-44740), Myd88 (sc-35987), lipoprotein lipase (LPL) (sc-44900), glycosylphosphatidylinositol high density binding protein 1 (GPI-HBP1) (sc-145686), clathrin (sc-35067), and caveolin-1 (sc-29241) were purchased from Santa Cruz Biotechnology. Scramble siRNA (sc-37007) used as control. Cells were transfected with the indicated siRNA duplex targeting constructs (50 nM) and HiPerFect Transfection Reagent (QIAGEN, Hilden, Germany). After incubation for 24 h, downregulation of target gene expression was evaluated by RT-PCR.

The CHO homogenate was adjusted to 40 % sucrose and centrifuged on a discontinuous sucrose gradient for 20 h at 4 °C using an SW 41 Ti rotor (Beckman) [28 (link), 29 (link)]. After centrifugation, the homogenate was fractionated into 12 fractions and subjected to western blotting using the following antibodies: N1660 (Nicastrin; 1/3000 in TBS containing 0.1 % Tween; Sigma), anti-Aph-1a C-term antibody (Aph-1; 1/1000 in TOYOBO Can Get Signal; Covance), anti-PS1–CTF antiserum (Presenilin 1 CTF; 1/3000 in TBS containing 0.1 % Tween; gifts from Drs. T. Iwatsubo and T. Tomita, The University of Tokyo), anti-Pen-2 antibody (Pen-2; 1/3000 in TBS containing 0.1 % Tween; a gift from Dr. A. Takashima, National Center for Geriatrics and Gerontology), anti-caveolin antibody (caveolin-1; 1/1000 in TBS containing 0.1 % Tween; Santa Cruz), and anti-flotillin antibody (flotillin-1; 1/1000 in TBS containing 0.1 % Tween; BD).

In order to study the uptake of exosomes by different cancerous or non-cancerous prostate cell lines (with distinct AR expression phenotypes), equal numbers of cells were seeded in four-well chamber slides (Lab-Tek II chamber slide with cover, Thermo Fisher scientific). Next, as previously reported [52 ] fresh CLU_GFP labelled exosomes were incubated with PC3 (AR−ve) and LNCaP (AR+ve) PCa cell lines as well as RWPE-1 representing a benign epithelial prostate cell line, for 12 h at 37°C and 5% CO₂. CLU_GFP tagged exosomes were isolated from a CLU_GFP stably overexpressing LNCaP cell line. After removal of media, cells were fixed with ice-cold MeOH/Acetone (3:1) for 10 minutes, and then washed in TBS buffer and permeabilized in 0.1% Triton X-100 in TBS for 15 minutes at room temperature (RT). Non-specific binding was avoided by blocking in odyssey solution for 30 minutes at RT. Primary purified mouse anti E-Cadherin (1:250 BD Transduction Laboratories™) or rabbit anti Caveolin-1 (1:250 Santa Cruz, CA) were diluted in blocking agent and incubated with cells for 1 hour at RT. Secondary antibody, Alexa Fluor^® 568 goat antimouse IgG or Alexa Fluor^® 555 Donkey Anti-Rabbit IgG (1:500, Invitrogen), was incubated with cells for 30 minutes at RT. Finally, as described above, all slide chambers were mounted and monitored using confocal microscopy (LSM 780 Ziess, Heidelberg, Germany).