Anti mouse igg hrp

Human α-thrombin and adenosine 5’-diphosphate sodium salt (ADP) were purchased from Sigma-Aldrich (St Louis, MO, USA). PAR1-activating peptide (PAR1-AP; TFLLR-NH₂) and PAR4-activating peptide (PAR4-AP; AYPGKF-NH₂) were synthesised at Monash Institute of Pharmaceutical Sciences (Melbourne, Australia) by Assoc Professor Philip Thompson. The mouse anti-P-selectin (FITC anti-mouse CD62P) and human anti-P-selectin (PE anti-human CD62P) antibodies were purchased from BD Biosciences (San Jose, CA, USA). PE anti-CD45.2, PE anti-CD41a (human) antibody and PE anti-CD41 antibody (mouse) were purchased from Abcam (Melbourne, Australia). The PE-anti-GPIb-tail and non-immune rabbit IgG isotype were a generous gift from Assoc Professor Robert Andrews (Monash University, Melbourne, Australia). The anti-PAR1 antibody (ATAP2), anti-PAR3 antibody (8E8), HRP-anti-actin (I-19) and HRP- anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Anti-RBX1 antibody (ab133565, 1:1000 dilution for immunoblotting and 1:100 for IHC) was purchased from Abcam. Anti-POLR2A antibody (sc-47701,1:5000 dilution for immunoblotting and 1:200 for IHC), anti-CUL1 antibody (sc-17775, 1:1000 dilution), anti-CUL2 antibody (sc-166506, 1:1000 dilution), anti-CUL4A/4B antibody (sc-377188, 1:1000 dilution), anti-CUL5 antibody (sc-373822, 1:1000 dilution) and anti-CUL7 (sc-53810, 1:1000 dilution) antibody were obtained from Santa Cruz. Anti-Ki67 antibody (#12075, 1:100 dilution), anti-cleaved Caspase-3 (Asp175) (#9664, 1:1000 dilution), Anti-SKP2 antibody (#2652, 1:1000 dilution), Anti-VHL antibody (#68547, 1:1000 dilution) and Anti-CUL3 (#2759, 1:1000 dilution) were purchased from Cell Signaling Technology. Anti-p53 (sc-126, 1:1000 dilution), anti-actin (sc-1616, 1:5000 dilution), HRP-anti-mouse IgG (sc-2055, 1:5000 dilution), HRP-mouse IgG kappa binding protein (sc-516102, 1:5000 dilution) and HRP-anti-rabbit IgG (sc-2054, 1:5000 dilution) were purchased from Santa Cruz.

For the analysis of protein expression, cell pellets were washed and resuspended in PBS. Cells were centrifuged and pellets were stored at −20 °C. Cell pellets were then lysed in Winman’s buffer containing 1% NP-40, 0.1 M Tris–HCl pH 8.0, 0.15 M NaCl, and 5 mM EDTA, complemented with protease inhibitor cocktail (Roche). The total protein content was quantified using the DC™ Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions. Protein lysate (20 µg) were loaded on 12% SDS-PAGE gel and transferred into a nitrocellulose membrane (GE Healthcare, Cleveland, OH, USA). The following primary antibodies were used: rabbit anti-PARP-1 (1:2000, sc-7150, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-Caspase 3 (1:2000, 05-654, Merck Millipore, Darmstadt, Germany), and goat anti-actin (1:2000, sc-1616, Santa Cruz Biotechnology). The corresponding secondary antibodies were: anti-rabbit IgG-HRP, anti-mouse IgG-HRP, or anti-goat IgG-HRP (1:2000, Santa Cruz Biotechnology). The Amersham™ ECL Western Blotting Detection Reagents (GE Healthcare), the High Performance Chemiluminescence Film (GE Healthcare), and the Kodak GBX developer and fixer (Sigma) were used for signal detection [35 (link),36 (link)].

Fucoidan from Fucus vesiculosus was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) as fucoidan. Fucoidan from Laminaria japonica was a gift from Hi-Q Marine Biotech International, Ltd. (Taiwan) as HiQ-fucoidan. PI, NAC, anti-actin (AC-74), anti-pERK1/2 (MAPK-YT) and anti-p21 (CP74) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Lipofectamine 2000 was purchased from Invitrogen (Grand Island, NY, USA). Anti-AKT, anti-p-AKT (S473) and anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (CA, USA). Anti-CHOP, anti-GRP78, anti-eIF2α, and anti-rabbit IgG-HRP were purchased from GeneTex, Inc. (Hsinchu, Taiwan). Anti-caspase3 (8G10), anti-p-PERK (T980; 16F8), anti-PERK (D11A8), anti-p-eIF2α (Ser51, 119A11), anti-TLR4 (D8L5W) and anti-ATF4 (D4B8) were purchased from Cell Signaling (Beverly, MA, USA).

Cell lysates were prepared by incubation with a lysis buffer (40 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.1% Nonidet-P40) supplemented with protease inhibitors. Then proteins were then subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL). Primary antibodies were used to detect the relevant protein, and β-actin was used as loading control. Blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies and proteins were visualized using enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL), according to the manufacturer's protocol. Secondary antibodies, anti-mouse IgG-HRP, anti-goat IgG-HRP and anti-rabbit IgG-HRP were purchased from Santa Cruz biotechnology (CA, USA).

Cells were washed in ice‐cold PBS and sedimented at 1000 g for 10 min at RT. Pellets were suspended in RIPA buffer (10 mm Tris/HCl, pH 7.4, 1% sodium deoxycholate, 1% Triton X‐100, 0.1% SDS) containing 1 mm PMSF and protease inhibitor cocktail (Sigma‐Aldrich). Protein concentration was quantified using Bicinchoninic acid (Thermo Scientific, Rockford, IL, USA). Total proteins were separated by SDS/PAGE and transferred to Porablot NCP membranes (Macherey‐Nagel, Düren, Germany). Blots were probed with anti‐FLAG (1 : 2000; Sigma‐Aldrich), anti‐E‐cadherin (1 : 2000; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1 : 2000; Cell Signaling Technology), anti‐Snail (1 : 2000; Cell Signaling Technology), and β‐actin (1 : 2000; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Primary antibody binding was detected with anti‐goat IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), anti‐mouse IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), or anti‐rabbit IgG‐HRP (1 : 2000; Santa Cruz Biotechnology). Membranes were revealed using the EZ‐ECL chemiluminescence kit (Biological Industries, Haemek, Israel) and the ChemiDoc Touch Gel Imaging System (Bio‐Rad, Hércules, CA, USA).