Anti eea1

Recombinant human TNF-α was purchased from R&D Systems (Wiesbaden-Norderstedt, Germany). Anti-PAR-1 (ATAP2), anti-NQO1, anti-eNOS, and anti-VCAM-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-phospho-HDAC5, anti-HDAC5, anti-phospho-ERK5, anti-ERK5, anti-phospho-AKT, anti-AKT, anti-phospho-AMPK, anti-AMPK, anti-phospho-eNOS, anti-EEA1, anti-phospho-Src, anti-phospho-FAK, anti-phospho-Erk1/2, anti-Erk1/2 and anti-VE-cadherin were from Cell Signaling Technology (Danvers, MA, USA). Anti-COX-2 was from Cayman Chemical Company (Ann Arvor, MI, USA). Anti-tubulin was from Sigma–Aldrich (St. Louis, MO, USA). Phenylephrine, Acetylcholine and U46619 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Pertussis toxin (PTX) was purchased from Gibco. Small interfering RNA against human PAR-1 was purchased from Santa Cruz Biotechnology (sc-36663, Santa Cruz, CA, USA). For PAR-1 silencing, HUVECs were transiently transfected with 100 pmol/L of control RNA or siRNA targeting human PAR-1 using Lipofectamine 2000 reagent (#11668-019, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A non-specific control siRNA from Bioneer was used as a negative control. HUVECs were harvested 48–72 hours after siRNA transfection, protein expressions were assessed by immunoblotting with antibodies and mRNA levels by quantitative real time RT-PCR (RT-qPCR).

Cells were plated on coverslips and were transfected with shRNA for 48 h. Cells were then fixed for 15 min in 10% formalin and permeabilized for 5 min with 0.3% Triton X-100. After blocking in 10% normal goat serum, cells were stained using an anti-E-cadherin antibody (R&D Systems; 1:50) followed by an Alexa546-conjugated secondary antibody. EGF-treated transfected cells were fixed/permeabilized for 10 min in methanol. After blocking in 1% BSA, the cells were incubated with an anti-EEA1 (Cell Signaling; 1:100) or anti-LAMP2 antibody (Abcam, Cambridge, UK; 1:100) followed by a FITC-conjugated secondary antibody and then with an Alexa555-conjugated anti-EGFR antibody (Cell Signaling; 1:100) Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) to visualize nuclei. Mounted samples were visualized with a confocal microscope (LSM 510 META; Carl Zeiss, Jena, Germany). Intensity of E-cadherin staining was quantitated by ImageJ software. Quantitative co-localization analysis of EGFR and EEA1 or LAMP2 was performed by calculating Pearson’s correlation coefficient (r) using ImageJ software.

Cells were grown on cover glasses for 1 day, rinsed with TBS at room temperature and fixed for 10 min with 4% paraformaldehyde. After rinsing with TBS, in the case of permeabilized conditions, cells were incubated with 0.1% Triton X-100 in TBS for 30 min followed by 30 min in blocking buffer (BSA 2% in TBS-T 0.1% Triton X-100) at room temperature. For non-permeabilized conditions, cells were directly incubated in blocking buffer (BSA 2% in TBS) for 1 h at room temperature. Next, cells were incubated in the suitable primary antibodies for 1 h at room temperature and rinsed repeatedly with TBS before incubating in the appropriate fluorescein-labelled secondary antibody for 1 h at room temperature. Cells were then washed extensively with TBS and mounted on a glass slide in Prolong Diamond Antifade mountant with DAPI (Pierce, Thermo Fisher Scientific). The following primary antibodies were used: anti-EEA1 (1:1000) (Cell Signalling), anti-LAMP-1 (1:1000) (Santa Cruz), anti-avb3 (1:50) clone LM609 (Merck), and anti-tubulin (1:1000) (DM1a-FTIC, Sigma). The following secondary antibodies were used: goat anti-mouse IgG (H + L) Alexa Fluor Plus 488 and goat anti-rabbit IgG (H + L) Alexa Fluor 633 (Thermo Fisher Scientific) (1:1000).

anti-H3K27ac (Cat# 8173), anti-Batf (Cat# 8638), anti-H3K27me3 (Cat# 9733), anti-Mbd3 (Cat# 99169), anti-Chd3 (Cat# 4241), anti-Rbap46 (Cat# 6882), anti-Hdac1 (Cat# 34589), anti-Hdac2 (Cat# 57156), anti-EEA1 (Cat# 3288), anti-H3 (Cat# 4499), and anti-β-actin (Cat# 3700) were from Cell Signaling Technology (Danvers, USA). Anti-Kcnt2 (Cat# bs-12177R) was from Bioss Antibodies (Beijing, China). Anti-CD127 (A7R34), anti-c-Kit (2B8), anti-CD3 (17A2), anti-CD4 (GK1.5), anti-CD19 (1D3), anti-NK1.1 (PK136), anti-CD150 (mShad150), anti-CD34 (RAM34), anti-CD45 (30-F11), anti-CD90 (HIS51), anti-Sca-1 (D7), anti-CD25 (PC61.5), anti-Flt3 (A2F10), anti-α₄β₇ (DATK32), anti-RORγt (AFKJS-9), anti-NKp46 (29A1.4), anti-Gata3 (TWAJ), anti-KLRG1 (2F1), anti-PLZF (Mags.21F7), Lineage cocktail (88-7772-72), anti-CD48 (HM48-1), Anti-IL-17 (eBio17B7), and anti-CD16/32 (93) were purchased from eBiosciences (San Diego, USA). Anti-BrdU (600-401-C29) was purchased from ThermoFisher. Paraformaldehyde (PFA) and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich. The IL-17 ELISA kit was purchased from eBiosciences.