Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 1. RT-PCR analysis of human leu- kocytes and endothelial cells for PAR RNA expression. Total RNA was ex- tracted from human eosinophils (A), neu- trophils (B), mononuclear cells (C), or en- dothelial cells (HUVECs; D) and was sub- ject to RT-PCR using random hexamer primers for the RT step and PAR-specific primers for the PCR step. PCR products were analyzed by agarose gel electro- phoresis and compared with a 100-bp lad- der (left lane). Expected product sizes were: PAR1, 300 bp; PAR2, 399 bp; PAR3, 372 bp; PAR4, 242 bp. Figure is a representative example of five different donors.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Random Hexamer, Agarose Gel Electrophoresis

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 3. Flow cytometry of human eosinophils for PAR1 and PAR2. Purified human eosinophils were stained with mAb to PAR1 (ATAP2, A and B) or PAR2 (SAM11, C and D). Cells were unfixed to study surface-only expression (A and C) or fixed and permeabilized to look at surface and intracellular levels (B and D). mAb are shown by the solid line, and appropriate isotype controls are represented by the dotted lines. (A–D) Representative example of four independent experiments. The levels of PAR1 and PAR2 in purified eosinophils or mixed granulocyte population following fixation and permeabilization were measured in control and asthmatic donors (each group, n4). (E) The results are shown as a ratio of the mean fluorescence intensity (MFI) of PAR mAb:isotype control.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Flow Cytometry, Staining, Expressing, Control

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 5. GAFS assay to measure shape change in response to PAR agonists in eosinophils (Eos) and neutrophils (Neuts). The GAFS assay was used to assess shape change by measuring increases in FSc of a mixed population of eosinophils and neu- trophils using flow cytometry. The eosinophil pop- ulation (R1) is distinguished from the neutrophils (R2) by their autofluorescent properties on the FL2 channel (A). Resting eosinophils and neutrophils (C and D) showed an increase in mean FSc when stimulated with PAF (E, P0.001; F, P0.001). (B) The mean FSc of resting and stimulated cells from a typical experiment is shown. Eotaxin was also used as a control to increase the mean FSc in eosinophils only. (G and H) The results shown are expressed as the percent increase in FSc induced by each agonist compared with buffer-stimulated cells after 5 and 30 min incubation (n6). Eosin- ophils (G) responded to the PAR1 peptide at both time points (5 min, P0.004; 30 min, P0.010) but not to thrombin. Activation via trypsin or PAR2 peptide (P0.008) was only seen after 30 min. Neutrophils (H) responded to trypsin after 30 min (P0.033) and at 5 and 30 min with peptides to PAR1 (5 min, P0.023; 30 min, P0.009) and PAR2 (5 min, P0.021; 30 min, P0.001). PAR4 was considered ineffective in this assay in either cell type.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Cytometry, Control, Incubation, Activation Assay

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 6. Release of cys-LT from human eosinophils in response to PAR activation. Purified human eosinophils were primed with 5 ng/ml IL-5 and treated with PAR agonists for 60 min. Cell-free supernatants were assayed for cys-LT using a commercially available EIA (n4–7). Release of cys-LT was very donor-variable, and only PAR2 peptide stimulated significant release compared with assay buffer alone (P0.0189). As a positive control, eosino- phils were treated with IgG-coupled sepharose beads, and release of over 4 ng/ml cys-LT was detected (data not shown).

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Activation Assay, Positive Control

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 7. Generation of ROS in human eosinophils in response to PAR activa- tion. Eosinophils were resuspended in assay buffer containing lucigenin and stimulated with PAR ligands and peptides. ROS generation was measured by chemiluminescence, and 1 M PAF was used as a positive control (P0.0041). Thrombin was inactive, whereas trypsin (P0.0062) or the PAR2 peptide (P0.0107) induced significant ROS generation. The PAR2 control peptide also stimulated some ROS generation, but this was significantly reduced compared with the active peptide (P0.0175).

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Positive Control, Control

Journal: PLoS ONE

Article Title: The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet α IIb β 3 Function

doi: 10.1371/journal.pone.0008304

Figure Lengend Snippet: ( A ) Lysate from resting washed platelets was evaluated by SDS-PAGE and immunoblotted with anti-PAR3 antibody (∼43 kDa). The blot was stripped and reprobed with anti- PAR4 (∼38 kDa) and then with anti-actin (loading) antibodies. Blots are representative of two experiments. Washed platelets were incubated with anti-JON/A PE antibody ( B ) or anti-α IIb FITC antibody ( C ) or anti-P-selectin antibody ( D ) and subjected to flow cytometry. Results are expressed as ±SEM of 5–6 experiments. Compared to thrombin stimulated WT platelets, the decreased binding of JON/A antibody to PP1cγ −/− platelets was significant at *p = 0.003.

Article Snippet: Antibodies to theα, and γ isoforms of PP1c, PAR3 and PAR4 were purchased from Santacruz Biotechnology (Santacruz, CA). β isoform of PP1c was from Upstate Biotechnology/Millipore (Billerica, MA).

Techniques: SDS Page, Incubation, Flow Cytometry, Binding Assay

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 expression level in lung cancer cells transfected either with pcDNA3-PAR2 or PAR2 shRNA. (A) PAR2 expression level after A549 cells were transfected with pcDNA3-PAR2; (B) PAR2 expression level after H1299 cells were transfected with pcDNA3-PAR2; (C) PAR2 expression level after A549 cells were transfected with PAR2 shRNA; (D) PAR2 expression level after H1299 cells were transfected with PAR2 shRNA.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, shRNA

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: Overexpression PAR2 promoted growth of lung cancer cells with or without paclitaxel. (A–D) The growth of A549 cells with or without paclitaxel after transfection with pcDNA3-PAR2; (E–H) The growth of H1299 cells with or without paclitaxel after transfection with pcDNA3-PAR2.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Transfection

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: Knockdown PAR2 decreased growth of lung cancer cells with or without paclitaxel. (A–D) The growth of A549 cells with or without paclitaxel after transfection with PAR2 shRNA; (E–H) The growth of H1299 cells with or without paclitaxel after transfection with PAR2 shRNA.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Transfection, shRNA

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 inhibited paclitaxel-associated apoptosis in lung cancer cells. (A) Caspase 3/7 activity in A549 cell transfected with pcDNA3-PAR2; (B) Caspase 3/7 activity in H1299 cell transfected with pcDNA3-PAR2; (C–F) Bcl-2 and BAX expression in A549 and H1299 cells.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Transfection, Expressing

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 played essential roles in invasion and migration of lung cancer cells. (A, B) up-regulation of PAR2 increased migration and invasion of A549 cells. (C, D) down-regulation of PAR2 decreaased migration and invasion of A549 cells. (E, F) up-regulation of PAR2 increased migration and invasion of H1299 cells. (G, H) down-regulation of PAR2 decreased migration and invasion of H1299 cells.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Migration

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 altered PTEN/AKT protein expression in lung cancer cells. (A–C) Impact of up-regulation of PAR2 on expression of p-AKT, AKT and PTEN in A549 and H1299 cells. (D–F) Impact of down-regulation of PAR2 on expression of p-AKT, AKT and PTEN in A549 and H1299 cells.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 expression in human lung cancer tissue. (A) PAR2 levels in different stage of lung cancer tissue and normal lung tissue. (B) Immunohistochemical studies for Ki-67 and PTEN on different levels of PAR2 in lung tissue.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Immunohistochemical staining