Mouse monoclonal antibodies

Immunolocalization of PARs on fibroblasts was performed using cells cultured on glass slides. The cells were rinsed with PBS and fixed in 4 % (v/v) paraformaldehyde in PBS for 30 min at RT. The cells were permeabilized with 0.2 % Triton X (v/v) in PBS for 5 min at RT, and rinsed in PBS. After blocking the cells in 20 % normal horse serum, the fibroblasts were incubated with antibodies raised against individual PARs, including mouse monoclonal antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) raised against PAR-1 (ATAP2 sc-13503) and PAR-2 (SAM11; sc-13504), as well as rabbit antibodies (commercially prepared by Chiron, Clayton, Australia) raised against PAR-3 (₃₇TLPIKTFRGAPPNSFEEFP₅₅) and PAR-4 (₂₈EDDSTPSLLPAPRGYPGQV₃₉). Equivalent dilutions of isotype-matched mouse and rabbit antibodies (Dako, Via Real Carpinteria, CV) were also used as appropriate controls. The primary antibodies were incubated overnight at 4°C. Following two washes in PBS, the cells were incubated with appropriate Alexa 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR) for 60 min at RT, rinsed twice in PBS, mounted in a nonfluorescent mounting medium, and kept in the dark until ready for viewing using fluorescence (TE2000-U; Nikon Corp., Chiyoda-ku, Tokyo, Japan) or confocal microscopy (Biomedical and Imaging Analysis Facility, UWA, Perth, WA, Australia).

The antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz), including mouse monoclonal antibodies against Rho A, MMP‐9, AKT1, β‐catenin, N‐cadherin, histone H3 lysine 4 (H3K4me3), proliferating cell‐nuclear antigen (PCNA) and β‐actin. The antibodies were respectively obtained by Abcam Technology (Abcam) and Cell Signalling Technology, including mouse/rabbit polyclonal antibodies against RhoA Ser188, AKT Thr180Tyr182 and NFκB p50 bought from all culture materials were obtained from Gibco (Grand Island, NY, USA). Protease inhibitor cocktails, reactive oxygen species (ROS) scavenger (N‐acetyl cysteine [NAC]), dihydroethidium (DHE), NFκB inhibitor (PDTC), SDS, RhoGTPase inhibitor (CCG‐1423), NP‐40, phosphoinositide 3‐kinase inhibitor (wortmannin), sodium deoxycholate, 2,7‐dichlorodihydrofluorescein diacetate (H2DCFDA) and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) were purchased from Sigma. Rabbit polyclonal antibodies against CHGA and UCHL1 were purchased from Bioss Inc.

For immunofluorescence assays, polarized epithelial cells were fixed with 4% paraformaldehyde and 2% sucrose in PBS for 5 min, and then permeabilized with 0.01% Triton X-100 in 4% paraformaldehyde for 5 min. For detection of EEA1 and rabankyrin, rabbit antibodies were used (both from Abcam) (1 μg/ml). LAMP1 and LBPA were detected using mouse monoclonal antibodies (Santa Cruz Biotechnology and Millipore, respectively) (1 μg/ml). For detection of HIV-1 p24, we used mouse and rabbit anti-p24 antibodies (NIH AIDS Research and Reference Reagent Program and Abcam) (5 μg/ml of each). For detection of CD45, mouse monoclonal antibodies were used (1.2 μg/ml) (R&D). Secondary antibodies labeled with DyLight 488, DyLight 594 and Alexa Fluor were purchased from Jackson ImmunoResearch. Cell nuclei were counterstained with TO-PRO-3 iodide or DAPI (blue) (Molecular Probes). The specificity of each antibody was confirmed by negative staining with the corresponding primary isotype control antibody. For detection of F-actin, cells were stained with fluorescence-labeled phalloidin (Thermo Fisher Scientific). Cells were analyzed by using a Leica SP5 laser confocal microscope (Leica Microsystems) or Nikon Eclipse E400 fluorescence microscope (Nikon).

Mouse monoclonal antibodies to N-cadherin (D-4), vimentin (V9), Snail (G-7), and the large extracellular loop of CD82 (G-2) and rabbit polyclonal antibodies to Slug (H-140), Twist (H-81), FAK (A-17), TM4SF2 (Y-19), phospho-ILK^(T173), and phospho-FAK^(Y925), and the C-terminus of CD82 (C-16) and goat polyclonal antibodies to integrin α3 (I-19), α5 (P-19), and α6 (N-19) subunits and an inhibitor to FAK (FAK inhibitor 14) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal Antibodies to integrin β₁ subunit (P5D2) and α-SMA (E184) and an inhibitor to ILK (Cpd22) were purchased from Millipore (Billerica, MA). Monoclonal antibodies to E-cadherin (36/E-cadherin) and fibronectin (C6F10) were obtained from BD Biosciences (San Diego, CA). Antibodies to phospho-Src^(Y416) and ILK were purchased from Cell Signaling (Beverly, MA) and Abcam (Cambridge, UK), respectively. Alexa fluor-conjugated goat anti-mouse (Alexa488) and goat anti-rabbit (Alexa555) secondary antibodies were from Life Technologies (Grand Island, NY). The small interfering RNAs (siRNAs) targeting integrin α₃, α₅, or α₆ subunit were obtained from Bioneer (Daejeon, Korea) and sequences are listed in the supplementary Table S2. All other reagents were from Sigma-Aldrich (St. Louis, Mo) unless indicated otherwise.