Ab2910

Expression of dynamin-2, caveolin-1 and clathrin after their downregulation by specific siRNA or in A549-shCLTC and A549-shCAV-1 clones was assessed by Western blotting analysis. Cells (3 × 10⁵ cells per sample) were lysed with Laemmli buffer heated to 95°C (150 μL/sample), scraped off the plate, sonicated and boiled (95°C, 3 min). Proteins were separated using 10% acrylamide gel by SDS-PAGE and transferred to nitrocellulose membrane (Amersham Protran, GE Healthcare, USA). The membrane was blocked with 5% (wt/vol) nonfat dry milk (Carl Roth, Germany) in Tris-Buffered Saline containing 1% Tween 20 and probed with the appropriate primary antibodies, followed by incubation appropriate horseradish peroxidase-conjugated IgG secondary antibody. Detection was performed with Pierce ECL Western Blotting Substrate (Thermo Fischer Scientific, USA), using ChemiDoc Imaging System (Bio-Rad, USA). Densitometry was performed with ImageJ software (NIH, USA). The following antibodies were used: anti CLTC (ab21679, Abcam, UK), anti CAV-1 (ab2910, Abcam, UK), anti DNM-2 (ab3457, Abcam, UK). Proteins were normalized to the total protein stained with amidoblack and the results were presented as relative expression of proteins compared to control cells.

Whole-protein extracts from cells or tumors were prepared after cell scrapping or tissue homogenization, respectively, in RIPA buffer and separated on SDS-PAGE gels (NuPAGE 4–12% Bis-Tris Protein Gels, Invitrogen). Membranes were probed using the following primary antibodies: rabbit anti-CAV1 1:500 (Abcam, ab2910), rabbit anti-HER2 1:800 (Abcam, ab131490), mouse anti β-actin 1:20,000 (Sigma, A1978), rabbit anti-ubiquitin 1:1,000 (Cell Signaling Technology, 3933 S), mouse anti-ERK 1:100 (Invitrogen, 14-9108-80), rabbit anti-pERK 1:500 (Invitrogen, 700012), rabbit anti-AKT 1:1,000 (Cell Signaling Technology, 9272 S), rabbit anti-pAKT, 1:2,000 (Cell Signaling Technology, 4060 S), rabbit anti-cleaved PARP, 1:1,000 (Cell Signaling Technology, 9541 S), rabbit anti-pHER2, 1:500 (Abcam, ab53290), rabbit anti-HER3, 1:500 (Abcam, ab32121), rabbit anti-pHER3, 1:2,500 (Abcam, ab76469), rabbit anti-EGFR 1:1,000 (Abcam, ab52894), rabbit anti-pEGFR 1:500 (Abcam, ab40815), mouse anti-pTyr 0.5 μg/mL (EMD Millipore, 05-321X), rabbit anti-CREB 1:1,000 (Cell Signaling Technology, 9197 S), rabbit anti-pCREB 1:1,000 (Cell Signaling Technology, 9198 S).
The membranes were then incubated with secondary antibodies IRDye^®800CW anti-rabbit or anti-mouse IgG 1:15,000 (LI-COR Biosciences) and imaged on the Odyssey Infrared Imaging System (LI-COR Biosciences) followed by densitometric analysis.

Primary antibody against PCAF (3305, CST), p53 (sc-126, santa cruz), p21 (sc-6246, santa cruz), p16 (sc-166760, santa cruz), Ubiquitin (sc-53509, santa cruz), phosphor-Histone H2A.X(Ser139) (#2577, CST), YAP (#14074, CST), MDM2 (BS-1223, Biogot), phospho-YAP (#13008, CST), TAZ (#83669, CST), phospho-TAZ (#59971, CST), clathrin (ab21679, abcam), caveolae (ab2910, abcam), mTOR (ab2732, abcam), p-mTOR (Ser2448) (ab109268, abcam), p-mTOR(Ser2481) (ab137133, abcam), IL-1β (sc-12742, snata cruz), IL-6 (sc-130326, santa cruz), GAPDH (#AP0063, Biogot), histone H3(BS3718, Biogot), and PA (P0500, Sigma-Aldrich) were purchased commercially.

HeLa cells were transfected with siRNA using Lipofectamine 2000 for 2×48 h or 1×72 h according to the manufactures’ recommendations. The siRNAs used were human-specific stealth siRNA (Invitrogen) for siRNA ctrl (negative control medium GC duplex), caveolin1 (HSS141467), Clathrin HC (HSS102017), GRAF1 (HSS118163) and cdc42 On target plus siRNA (J-005057-05) (Dharmacon). The cells were seeded on coverslips 24 h before optimal knockdown for PI assay. The efficiency of siRNA-based knockdown of endocytic proteins was determined by western blot. Primary antibodies used were mouse anti-GAPDH 1:5000 (MAB374, Millipore), rabbit anti-caveolin1 1:10,000 (ab2910, Abcam), mouse anti-Clathrin HC 1:1000 (clone 23 610499, BD Biosciences), rabbit anti-GRAF1 (Ra83) (Lundmark et al., 2008 (link)) and rabbit anti-cdc42 1:1000 (ab109553, Abcam). Secondary antibodies, donkey anti-mouse and donkey anti-rabbit IgG coupled to IRDye 680LT or 800CW (Li-Cor Biosciences) were used for fluorescent detection using an Odyssey Sa instrument (Li-Cor Biosciences). For detection with ECL Prime (GE Healthcare) anti-rabbit and anti-mouse IgG conjugated to horseradish peroxidase (HRP) from Agrisera or Sigma-Aldrich were used. The level of knockdown was quantified using Image Studio (Li-Cor Biosciences) for fluorescent images and ImageJ for films (Schneider et al., 2012 (link)).

Western blot was used to detect common EV marker proteins and FN1. 10–20 μg protein from EV or cell lysates were subjected to sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). Blots were incubated with primary antibodies to CD63 (1:100; MilliporeSigma, Burlington, MA, United States), flotillin-1 (1:200; BD Biosciences, San Jose, CA, United States), CD9 (1:500; Abcam, Cambridge, MA, United States), HNF4α (1:200, Thermo Fisher Scientific, Waltham, MA, United States), clusterin (Clu, 1:500; Proteintech, Rosemont, IL, United States), major vault protein (MVP; 1:500; Proteintech, Rosemont, IL, United States), albumin (1:500, Abcam, United States), mFN1 (1:500, Abcam, United States), huFN1 (1:500, Sinobiological, Beijing, China), or β-actin (1:1,000; Invitrogen, Carlsbad, CA, United States). Blots were developed using an Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, United States). CLTC (ab21679, Abcam), CAV1 (ab2910, Abcam), and DNM2 (A303-513A, Bethyl Laboratories, Montgomery, TX, United States) antibodies were used to measure the knockdown efficiency of their respective targets in AML12 or passaged mHSC transduced with lentiviral short-hairpin RNA.

(RS)-3,5-DHPG and dynasore were purchased from Tocris Bioscience (Bristol, UK). AP5 (D-2-amino-5-phosphonopentanoate), MPEP, cholesterol, and methyl-β-cyclodextrin (Mβ-CD) were obtained from Sigma (St. Louis, MO, USA). We used the following antibodies: anti-FMRP (MAB2160; Millipore, Billerica, MA, USA), anti-Cav1 (ab2910, Abcam, Cambridge, UK), anti-GluA1 (ab31232; Abcam), anti-GluA2 (ab20673; Abcam), anti-pan-cadherin (C1821; Sigma), anti-ERK1/2 (sc514302; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pERK1/2 (sc81492; Santa Cruz Biotechnology), anti-mTOR (sc517464; Santa Cruz Biotechnology), anti-pmTOR (sc293133; Santa Cruz Biotechnology), and anti-β-actin (A5316; Sigma).

For microscopy images, HeLa and C2C12 cells were cultured the day prior to the experiment on round-glass coverslips in a 24-well plate or a 96-well glass bottom plate and incubated with medium supplemented with the indicated concentrations of peptide/protein at 37 °C under 5% CO₂. Cells were washed three times with cold PBS, followed by a cleansing process utilizing light shaking with three washes of PBS, then three washes with 25 mM of Hepes to eliminate nonspecific protein binding. The cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min and washed three times with PBS to remove the PFA. All samples were stained with 1 mg/mL 4′,6-diamidino-2-phenylindole (DAPI Staining Solution ab228549, Abcam). Cav-1 (ab2910, Abcam), β-tubulin (05-661, Merck). Images were taken directly from the 96-well plate glass bottom, while coverslips from the 24-well plate were transferred into a glass slide and placed on the plate with sodium phosphate buffer mounting medium (25% Mowiol, 50% glycerol, 25% sodium base pH 8.0). Images were taken using a ZEISS LSM 900 in confocal mode, utilizing Zen 3.1 software. Fluorescence readouts were obtained for blue, green, bright red and far-red at ex/em of 358/461 nm, 495/519 nm, 561/594 nm, and 652/668 nm, respectively.