Journal: Cell reports

Article Title: Cdc42 activity in Sertoli cells is essential for maintenance of spermatogenesis

doi: 10.1016/j.celrep.2021.109885

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-PARD3 (Par3) , Novus , Cat#NBP1-88861; RRID: AB_11056253.

Techniques: Recombinant, Microarray, Real-time Polymerase Chain Reaction, Software

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black circle), PAR3 −/− (gray circle), and PAR3 +/− (white square) platelets were activated with the indicated concentrations of: ( A ) thrombin, (0.001–100 nM, ( B ) AYPGKF (0–2 mM), ( C ) convulxin (0.01–100 nM), or 20 µM of ADP for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques:

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Flow cytometric analysis of PAR4 expression in wild type (WT) (black line), PAR3 −/− (gray line), and PAR4 −/− (shaded) mice platelets using anti-PAR4-FITC antibodies.

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black) and PAR3 −/− (gray) platelets were incubated at 37°C for 5 min in the absence or the presence of 100 µM 2MeSAMP. After treatment, platelets were activated 100 nM thrombin ( A ,) or 2 mM AYPGKF ( B ) for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black line) and PAR3 −/− (gray line) platelets were resuspended in Ca 2+ -free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: ( A ) thrombin (1–100 nM), ( C ) AYPGKF (0.15–2 mM), or ( E ) 3 µM thapsigargin (TG). Quantitation of the change in peak Ca 2+ mobilization in platelets stimulated with: ( B ) thrombin, ( D ) AYPGKF, or ( F ) thapsigargin. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Quantitation Assay

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: The HEK293 cells were transfected with: ( A ) PAR4-Luc (1 µg) and PAR3-GFP (0–2.5 µg), ( B ) PAR3-Luc (1 µg) and PAR3-GFP (0–2.5 µg), or ( C ) PAR4-Luc (1 µg) and PAR4-GFP (0–2.5 µg). As a control experiment, the HEK293 cells were transfected with: ( D ) PAR3-Luc (1 µg) and rho-GFP (0–0.12 µg), or ( E ) PAR4-Luc (1 µg) and rho-GFP (0–0.12 µg). Forty-eight hours post-transfection, the cells were analyzed for GFP expression, Luc expression, and BRET. The curves were plotted as the ratio of GFP to Luc and all points from 3–6 independent experiments were analyzed by global fit to a hyperbolic or linear curve. The surface expression of PAR3 and PAR4 in the HEK293 cells was determined by flow cytometry. ( G ) V5-PAR4-GFP and V5-PAR3-GFP were detected with a V5 tag antibody conjugated to Alexa Fluor 647. ( H ) HA-PAR4-LUC and HA-PAR3-LUC were detected with a HA tag antibody conjugated to Alexa Fluor 647. The results are the mean (± SD) of two independent experiments. The number of PAR4 and PAR3 molecules on the HEK293 cells surface is calculated from the V5 or HA antibody standard curve using quantitative flow cytometry ( F ).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Transfection, Control, Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Targeting PKCθ Promotes Satellite Cell Self-Renewal

doi: 10.3390/ijms21072419

Figure Lengend Snippet: Lack of PKCθ stimulates symmetric self-renewal by regulating Pard3 polarization. ( A ): Representative pictures of single myofibers isolated from EDL muscles of WT and PKCθ-/- mice, after 48h in culture. Myofibers were stained for Pax7 (red) and MyoD (green), nuclei were counterstained with Topro3. ( B ): Quantification of symmetric division events. ( C ): Quantification of Pax7 + /MyoD − cell doublets, and ( D ): quantification of total Pax7 + /MyoD − cells in WT and PKCθ-/- single myofibers. ( E ): Representative pictures of single myofibers isolated from EDL muscles of WT and PKCθ-/- mice, after 36h in culture. Myofibers were stained for Pax7 (red) and Pard3 (green), nuclei were counterstained with Topro3. ( F ): Percentage ofSCs showing symmetric, low or asymmetric Pard3 distribution in WT and PKCθ-/- myofibers. ( G ): Percentage of SCs showing symmetric and asymmetric Pard3 distribution (WT, n = 3 mice, PKCθ-/-, n = 3 mice, n > 20 myofibers analyzed per mouse). Error bars represent mean ± sem, * p < 0.05 calculated by Student’s t -test.

Article Snippet: Primary antibodies mouse anti Pax7, rabbit anti-MyoD (1:50 Santa Cruz C20: sc-304, Dallas, TX, USA), and rabbit anti Pard3 (07-330 Merck Millipore, Burlington, MA, USA) were incubated O/N at 4 °C.

Techniques: Isolation, Staining