Journal: Journal of receptor and signal transduction research

Article Title: Evidence for Prostacyclin and cAMP Upregulation by Bradykinin and Insulin-Like Growth Factor 1 in Vascular Smooth Muscle Cells

doi: 10.3109/10799890903563768

Figure Lengend Snippet: VSMC were stimulated with either BK (10−8M) or IGF-1 (10−8M) for 10 min in the presence and absence of Src kinase inhibitor PP1 (10 μM). MAPK phosphorylation (p42mapk and p44mapk) were measured by immunoblot using anti-phosphotyrosine-MAPK antibodies (p-MAPK) and total MAPK was measured in the same immunoblot by stripping the membrane and re-immunoblotting with anti-total MAPK antibodies (t-MAPK). Release of 6-keto-PGF1α and cAMP into the media, were measured by RIA. Data are expressed as mean±SE and the bar graphs are representative of 6–8 separate experiments. * P<0.05 vs. control, †P<0.05 vs. BK, #P<0.05 vs. IGF-1.

Article Snippet: To demonstrate a role for Src kinases in ERK activation by BK and IGF-1, VSMC were pretreated for 40 min with a specific cell permeable inhibitor of Src family tyrosine kinases PP1 (10 μM, Biomol Research Laboratories Inc., Plymouth, PA) followed by stimulation with BK (10 −8 M) or IGF-1 (10 −8 M) for 10 min.

Techniques: Western Blot, Stripping Membranes

Journal: Journal of Cellular and Molecular Medicine

Article Title: PAI1: a novel PP1‐interacting protein that mediates human plasma's anti‐apoptotic effect in endothelial cells

doi: 10.1111/jcmm.13127

Figure Lengend Snippet: Human plasma decreases the interaction of PP1 with BAD in endothelial cells. ECs treated with different concentrations of HP according to Scheme B were subjected to immunoprecipitation–immunoblotting. IP: immunoprecipitation; IB: immunoblotting; WCL: whole‐cell lysate.

Article Snippet: Antibodies against BAD, 14‐3‐3, Akt, PP1 (recognizing all isoforms), GAPDH and an isotype‐matched IgG control for immunoprecipitation were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Clinical Proteomics, Immunoprecipitation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: PAI1: a novel PP1‐interacting protein that mediates human plasma's anti‐apoptotic effect in endothelial cells

doi: 10.1111/jcmm.13127

Figure Lengend Snippet: Identification of PAI1 as a PP1‐interacting protein. ( A ) HCMECs were treated with or without HP for 30 min. according to Scheme B and whole‐cell lysates subjected to immunoprecipitation using an anti‐PP1 antibody followed by SDS‐PAGE separation and silver staining. The positions of molecular mass markers are marked on the left. Band X is the gel band of interest which was subjected to LC‐MS/MS analysis. ( B ) HCMECs were treated with HP for 30 min. and whole‐cell lysates were prepared and subjected to immunoprecipitation–immunoblotting. ( C ) Whole‐cell lysates prepared from HCMECs treated with or without HP were incubated with an equal molar amount of GST‐tagged PP1 isoforms followed by pull‐down with glutathione‐sepharose beads. The eluted proteins were resolved by SDS‐PAGE followed by immunoblotting. Ig, immunoglobulin; IP, immunoprecipitation; IB: immunoblotting; WCL: whole‐cell lysate.

Article Snippet: Antibodies against BAD, 14‐3‐3, Akt, PP1 (recognizing all isoforms), GAPDH and an isotype‐matched IgG control for immunoprecipitation were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: PAI1: a novel PP1‐interacting protein that mediates human plasma's anti‐apoptotic effect in endothelial cells

doi: 10.1111/jcmm.13127

Figure Lengend Snippet: Schematic representation of the role of PAI1‐PP1 interaction in the regulation of BAD phosphorylation and the anti‐apoptotic effect in endothelial cells. ( A ) Under stress conditions such as hypoxia–reoxygenation and serum deprivation, PP1 binds to BAD, rendering BAD in an unphosphorylated state and the cell eventually undergoes apoptosis. ( B ) HP induces BAD phosphorylation and promotes EC survival through promoting the interaction between PAI1 and PP1 and displacing PP1 from complexing with BAD. Activation of Akt signalling also contributes to the phosphorylation of BAD in this process.

Article Snippet: Antibodies against BAD, 14‐3‐3, Akt, PP1 (recognizing all isoforms), GAPDH and an isotype‐matched IgG control for immunoprecipitation were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: PAI1: a novel PP1‐interacting protein that mediates human plasma's anti‐apoptotic effect in endothelial cells

doi: 10.1111/jcmm.13127

Figure Lengend Snippet: Identification of HP‐induced PP1‐interacting proteins in endothelial cells

Article Snippet: Antibodies against BAD, 14‐3‐3, Akt, PP1 (recognizing all isoforms), GAPDH and an isotype‐matched IgG control for immunoprecipitation were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Protease Inhibitor, Protein Binding, Ubiquitin Proteomics, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: HMGB1 Regulates Adipocyte Lipolysis via Caveolin-1 Signaling: Implications for Metabolic and Cardiovascular Diseases

doi: 10.3390/ijms26094222

Figure Lengend Snippet: Role of c-Src/CAV1 signaling in HMGB1 secretion from 3T3-L1 adipocytes. ( A ) Western blot analysis of CAV1, phosphorylated CAV1 (pCAV1), and α-tubulin during 3T3-L1 preadipocyte differentiation. ( B ) Quantitative results of panel ( A ). ( C ) Immunoprecipitation of cell lysates from undifferentiated and 5-day differentiated 3T3-L1 adipocytes with an anti-HMGB1 antibody, followed by immunoblotting with anti-pCAV1, CAV1, and HMGB1 antibodies. ( D ) Quantitative results of panel ( C ). ( E ) Undifferentiated and 5-day differentiated 3T3-L1 adipocytes were treated with increasing concentrations of PP1 (0, 0.1, 1, and 10 µM), a Src kinase inhibitor, for 24 h. Western blot analysis was performed to assess pCAV1 and α-tubulin, as well as secreted HMGB1 (sHMGB1) in the culture medium. ( F ) Quantitative results of panel ( E ). All data are presented as mean ± SEM from four independent experiments. * p < 0.05 versus the 0 min time point or vehicle-treated group. # p < 0.05 versus the untreated group (without PP1).

Article Snippet: PP1 (an inhibitor of Src-family tyrosine kinases) was from Tocris Bioscience (Bristol, UK).

Techniques: Western Blot, Immunoprecipitation