Anti upa

Whole-cell lysates were prepared using RIPA buffer, as described previously^{15 (link)}, and analyzed using the following primary antibodies: anti-Sp1, anti-Sp3, anti-uPA, and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-myc (Upstate Biotechnology, Lake Placid, NY, USA); anti-phospho-c-Jun(S63), anti-c-Jun, anti-phospho-ATF-2(T71), anti-ATF-2, anti-phospho-JNK(T183/Y185), anti-JNK, anti-phospho-Akt, anti-Akt, anti-survivin, anti-bcl-2, anti-phospho-IKKα/β(S176/180), anti-phospho-IκBα(S32), anti-phospho-NF-κB p65(S536), anti-cleaved caspase-3, and anti-PARP (Cell Signaling Tech., Danvers, MA, USA); anti-integrin α5 (BD Biosciences, San Jose, CA, USA); and anti-TMPRSS4 (in-house)^{15 (link)}. Where indicated, cells were treated with 0.1~10 μM KRT1853^{14 (link)} or IMD-0354 (Sigma, St Louis, MO, USA), 20~50 μM oxaliplatin (Sigma), or 0.1% dimethyl sulfoxide (DMSO) for 24~48 h before lysate preparation.

Western blot analyses were performed as described in detailed previously [23 (link)] with slightly modifications. CnAOEC and CnAOSMC previously treated with 1μg/ml of rDiACT or rDiFBAL for 24 h were lysed in ice-cold lysis buffer (20mM Tris—HCl (pH 7.5), 140mM NaCl, 10mM ethylendiaminetetraacetic acid, 10% glycerol, 1% Igepal CA-630, aprotinin, pepstatin, and leupeptin at 1μg/ml each, 1mM phenylmethylsulfonyl fluoride, and 1mM sodium orthovanadate). Non-stimulated cells were used as controls under the same conditions. Protein samples (10 μg) were separated by SDS-PAGE under reducing conditions and electrotransferred onto polyvinylidine difluoride membranes. Then, membranes were blocked before incubation with the following primary rabbit polyclonal antibodies: anti-tPA and anti-uPA (Santa Cruz Biotechnology Inc) according to the manufacturer's recommendations. After incubation with HRP-conjugated anti-rabbit secondary antibodies, bands were visualized by a luminol-based detection system with p-iodophenol enhancement. Anti-α-tubulin antibody (Oncogene Research Products) was used as control to confirm loading of comparable amount of protein in each lane. Protein expression was quantified by densitometry using the PDQuest Software v.8.0.1 (Bio-Rad).

Experimental animal sheep, heparin, propofol, isoflurane, potassium chloride, and whole pig blood were provided by Fuwai Hospital, Chinese Academy of Medical Sciences. Whole blood was collected from healthy animals using a venipuncture and stored in blood bag containing sodium citrate to prevent coagulation. 3’,3’‐dihexyloxacarbocyanine iodide (DiOC6) and FITC labeled human fibrinogen were purchased from Thermo Fisher Scientific (Waltham, MA, USA). RGDS (# 0 401 001 3130) and RGDS‐5‐TAMRA (5‐Carboxytetramethylrhodamine) peptide were obtained from Jiangsu Qiangyao Biological Technology (Co. Ltd. Jiangsu, China). SonoVue (Bracco Suisse SA.) was a commercially available clinical ultrasound contrast agent. Recombinant Human Urokinase Pro (PUK, # 20201102) was purchased from Tasly Pharmaceutical (Co. Ltd. Shanghai, China). Ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. 10% formalin, 2.5% glutaraldehyde solution and Triton‐X 100 were from Beijing Solarbio Science & Technology Co., Ltd. Phosphate‐buffered saline (PBS, 1×) is obtained from HyClone. The primary antibodies anti‐uPA (# sc‐59727) was purchased from Santa Cruz Biotechnology. The goat antimouse Alexa Fluor 488‐conjugated (# ab150113) was from Abcam.

Whole-cell lysates were prepared using RIPA buffer as described previously [19 (link)] and analyzed using the following primary antibodies: anti-uPA, anti-c-Src, anti-ZEB1, anti-β-actin, and anti-GAPDH (Santa Cruz Biotechnology); anti-vimentin (Sigma, St Louis, MO, USA); anti-myc (Upstate Biotechnology, Lake Placid, NY, USA); anti-Slug, anti-Snail, anti-cyclin D1, anti-phospho-c-Jun(S63), anti-c-Jun, anti-phospho-ATF-2(T71), anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-ERK1/2, anti-phospho-c-Src(Y416), anti-phospho-JNK(T183/Y185), and anti-JNK (Cell Signaling Tech., Danvers, MA, USA); anti-Twist1 (Abcam, Cambridge, MA, USA); anti-ZEB2 (Active Motif, Tokyo, Japan); anti-Axl (R&D systems, Minneapolis, MN, USA); anti-TMPRSS4 (in-house) [19 (link)]. Where indicated, cells were transiently transfected for 24 h and then treated with 40 mM PD98059, 15 mM SP600125, 3 mM SU6656 (Sigma), or 0.4% dimethyl sulfoxide (DMSO) for 24 h before lysate preparation.

Western blot analyses were performed as previously described [21 (link)] with slight modifications. CnAOEC and CnAOSMC previously treated with 1 μg/ml of DiES for 24 h were lysed in ice-cold lysis buffer (20 mM Tris–HCl (pH 7.5), 140 mM NaCl, 10 mM ethylendiaminetetraacetic acid, 10% glycerol, 1% Igepal CA-630, aprotinin, pepstatin, and leupeptin at 1 μg/ml each, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate). Non-stimulated cells were used as controls under the same conditions. Protein samples (10 μg) were separated by SDS-PAGE under reducing conditions and blotted onto polyvinylidine difluoride membranes. Membranes were blocked before incubation with primary antibodies: anti-tPA, anti-uPA, anti-Annexin A2 and anti-PAI-1 (Santa Cruz Biotechnology Inc) according to the manufacturer’s recommendations. After incubation with HRP-conjugated secondary antibodies, bands were visualized by a luminol-based detection system with p-iodophenol enhancement. Anti-α-tubulin antibody (Oncogene Research Products) was used to confirm loading of comparable amount of protein in each lane. Protein expression was quantified by densitometry using Scion Image Software (Scion).

The thrombi, which experience 15 min IST, were fixed in 4% paraformaldehyde for 24 h. The fixed thrombus was embedded in paraffin and then sectioned and dewaxed. All procedures were performed in strict accordance with the tissue section preparation specifications. The histology sections are made along the sagittal plane with a thickness of 8 µm. After blocking with goat serum containing 0.3% Triton‐X 100 for 1 h at room temperature, the slides were incubated with primary antibodies overnight at 4 °C. After washing three times with PBS, the slides are incubated with fluorescence‐labeled secondary antibodies for 1 h at room temperature in the dark. Slices are washed three times in PBS, and then counterstained with Fluorescent mounting medium (ZLI‐9556; ZSGB‐Bio). The primary antibodies are as follows: anti‐uPA (1:00; sc‐59727, Santa Cruz Biotechnology). The secondary antibodies are conjugated to goat anti‐mouse Alexa Fluor 488‐conjugated (1:400; ab150113, Abcam). Fluorescence of PUK is observed under a Laser Confocal Microscope Nikon A1 (Nikon, Japan).

The cells were lysed in NP-40 lysis buffer containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche, Rotkreuz, Switzerland). After separation using SDS–PAGE, the proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, USA). Subsequently, the membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 hour at room temperature. The blots were probed with the relevant primary antibodies overnight at 4°C, washed, and probed with a species-specific horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Beverly, USA). An enhanced chemiluminescence detection method (Pierce ECL Western Blotting Substrate, Thermol, Beverly, MA, USA) was used to visualize the blots. The primary antibodies used were anti-EMMPRIN, anti-MMP-2, anti-uPA, anti-Cathepsin B (Santa Cruz Biotechnology, USA), and anti-β-actin (Sigma–Aldrich, MO, USA).