Journal: PLoS ONE

Article Title: Loss of Protein Kinase C-δ Protects against LPS-Induced Osteolysis Owing to an Intrinsic Defect in Osteoclastic Bone Resorption

doi: 10.1371/journal.pone.0070815

Figure Lengend Snippet: WT and PKC-δ KO BMMs were serum-starved overnight before stimulation with M-CSF and 100 ng/ml of RANKL at the indicated times. (A) Western blot analysis of total protein from WT and PKC-δ KO BMMs stimulated with M-CSF and 100 ng/ml of RANKL (short time-course, 0–120 min). (B) Quantitative analysis of short-term ERK phosphorylation relative to total ERK protein expression by densitometry of western blot images. (C) Western blot analysis of RANKL-induced osteoclastogenesis (long time-course, 0–6 days). (D) Quantitative analysis of Src Tyr-416 and Tyr-527 phosphorylation status relative to total Src protein expression, and long-term ERK phosphorylation relative to total ERK protein expression, as measured by densitometry of western blot images. β-actin was probed as a loading control. Statistical analysis was performed by comparing to WT in each time point. *, p-value <0.05. **, p-value<0.01.

Article Snippet: Antibodies for western blotting were purchased from the following sources: mouse IκB-α (C-21), Phospho-ERK1/2 (E-4) (Santa Cruz Biotechnology Inc.); mouse JNK (#9252), Phospho-JNK (#9251), p38 (#9212), Phospho-p38 (#9211), PKC-δ (#2058), Phospho Thr-505 PKC-δ, Phospho-PKC(pan)(#9371), Phospho Tyr-416 Src family (#2101), Phospho-MARCKS (#8722) (Cell Signaling); mouse NFATc1 (556602) (BD Pharmingen); mouse ERK1/2 (V114a) (Promega Corp.); mouse beta-actin (JLA20) (Calbiochem); mouse anti-c-Src antibody was a gift by A/Prof Heung-Chin Cheng, Australia; conjugated peroxidase IgG antibodies and mouse anti-α-Tubulin (T3526) were purchased from Sigma-Aldrich.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: 17β-Estradiol Enhances Breast Cancer Cell Motility and Invasion via Extra-Nuclear Activation of Actin-Binding Protein Ezrin

doi: 10.1371/journal.pone.0022439

Figure Lengend Snippet: (A) T47-D cells were exposed to 10 −8 M E2 for 15 min, in the presence or absence of the pure ER antagonist ICI 182,780 (ICI - 1 µM), of the MEK inhibitor PD98059 (PD - 5 µM), of the G protein inhibitor, PTX (100 ng/mL), of the PI3K inhibitor wortmannin (WM - 30 nM), of the ROCK-2 inhibitor, Y-27632 (Y - 10 µM) or of the c-Src kinase inhibitor, PP2 (10 µM). Cell content of wild-type or phosphorylated ezrin are shown. P-ezrin densitometry values were adjusted to ezrin intensity and then normalized to expression from the control sample. ** = P<0.01 vs. control, # = P<0.01 vs. E2. (B) and (C) show the time- and dose-dependent c-Src activation in T47-D breast cancer cells after treatment with E2. Total cell amount of wild-type (c-Src) or Tyr 416 -phosphorylated c-Src (P-c-Src) are shown with western blot. P-c-Src densitometry values were adjusted to c-Src intensity, then normalized to expression from the control sample. (D) T47-D cells were exposed to 10 −8 M E2 for 15 min after transfection with 100 nM c-Src siRNAs or control scrambled siRNAs for 48 h. Total actin, c-Src, ezrin, P-ezrin, Akt and P-Akt amounts and statistics for densitometry are shown. ** = P<0.01 vs. corresponding control. (E) T47-D cells were treated with 10 −8 M E2 for 15 min, with or without the ER antagonist ICI 182,780 (ICI - 1 µM), PI3K inhibitor wortmannin (WM - 30 nM) or c-Src kinase inhibitor, PP2 (10 µM). Active Akt (P-Akt) densitometry values were adjusted to wild-type Akt intensity, then normalized to expression from the control sample. ** = P<0.01 vs. control. # = P<0.01 vs. P. (F) Cells were exposed to 10 nM E2 for 15 min after transfection with wild type p85α (WT p85α, 1.5 µg) or dominant-negative p85α (Δp85α, 1.5 µg) for 48 h. Cell contents of actin, p85 protein, wild-type and phosphorylated ezrin and statistics for densitometry are shown. ** = P<0.01 vs. corresponding control without transfection. All these experiments were performed in triplicates and representative images are shown.

Article Snippet: Antibodies used were: phospho-Thr 567 -Ezrin (A00334, Genescript, Aachen, Germany), ezrin (#3145 ,Cell signaling), phospho- Tyr 416 -Src (# 2101, Cell Signalling Technology), Src (# 2108, Cell signaling) , ROCK-2 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphor- Ser 473 - Akt (Upstate Biotechnology, Inc., Lake Placid, NY), actin (sc-1615, Santa Cruz), Akt (# 9272, Cell Signalling Technology).

Techniques: Expressing, Activation Assay, Western Blot, Transfection, Dominant Negative Mutation

Journal: Cells

Article Title: Na,K-ATPase Acts as a Beta-Amyloid Receptor Triggering Src Kinase Activation

doi: 10.3390/cells11172753

Figure Lengend Snippet: Aβ 42 activates Src-kinase in nanomolar concentrations. ( A ) Co-localization studies with Proximity Ligation Assay. Close proximity of Na,K-ATPase α1-subunit and Src kinase in the SH-SY5Y neuroblastoma cells. The confocal merged image of Hoechst fluorescence (blue), RNASelect (green) and Duolink Red (red) fluorescence is presented. Scale bar—50 µm. ( B ) The effect of Aβ 42 on the Na,K-ATPase transport activity in SH-SY5Y cells. K + (Rb + ) influx after 30 min treatment with 100 nM Aβ 42 . Total Rb + influx into the cells was measured in the absence of ouabain (Total); “Passive” denotes ouabain-resistant component of Rb + influx in the sample where ouabain was added. Difference between the total and the passive fluxes gives the active (Active) Rb + influx mediated by the Na,K-ATPase. ( C ) The changes in Na,K-ATPase levels in SH-SY5Y neuroblastoma cells after 30 or 60 min of incubation with 100 nM Aβ 42 . Na,K-ATPase levels were evaluated by flow cytometry. ( D , E ) Dose-dependent activation of Src by Aβ 42 . The ratio of phospho(Tyr)-416 Src to the total Src has been calculated. The phosphorylated and total Src levels have been measured with Western blot in SH-SY5Y neuroblastoma cells treated with 100 nM, 500 nM and 2 µM of Aβ 42 for 30 min and normalized for control. Mean values ± SD from at least three independent experiments are shown. *— p < 0.05, ***— p < 0.001 compared to the control, ns—nonsignificant.

Article Snippet: The membrane was blocked in 5% nonfat milk in TBST (50 mM Tis-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20), and incubated with primary rabbit antibodies to phospho (Tyr 416)-Src kinase (p-Src; Cell Signaling Technology, Danvers, MA, USA, 6943S) or total Src kinase (Cell Signaling Technology, Danvers, MA, USA, 2108S) in TBST overnight at +4 °C.

Techniques: Proximity Ligation Assay, Fluorescence, Activity Assay, Incubation, Flow Cytometry, Activation Assay, Western Blot

Journal: Cells

Article Title: Na,K-ATPase Acts as a Beta-Amyloid Receptor Triggering Src Kinase Activation

doi: 10.3390/cells11172753

Figure Lengend Snippet: Modeling of Src kinase interaction with Na,K-ATPase. Interaction interface between human Na,K-ATPase and Src kinase studied by molecular modeling. ( A ) Contact frequency histogram of Na,K-ATPase residues according to 70 complexes of Na,K-ATPase:Src kinase obtained by targeted docking on servers PatchDock and Haddock. ( B ) Best rated docking complex after 100 ns of MD. ( C ) Interaction interface between Na,K-ATPase and Src kinase in the best rated docking complex after 100 ns of MD. Src kinase is colored cyan and Na,K-ATPase is colored gray. The interaction surface is shown with translucent pink. Na,K-ATPase residues 400–418 that interact with Src kinase are colored with magenta and red (residue 410–418 are the part of NaKtide peptide sequence). Residues 419–429 of NaKtide peptide sequence that do not participate in interaction with Src kinase are colored beige. Cysteines 454, 458 and 459 that are located inside the interaction are colored yellow. Tyrosine 416 which is located inside the interaction interface is shown.

Article Snippet: The membrane was blocked in 5% nonfat milk in TBST (50 mM Tis-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20), and incubated with primary rabbit antibodies to phospho (Tyr 416)-Src kinase (p-Src; Cell Signaling Technology, Danvers, MA, USA, 6943S) or total Src kinase (Cell Signaling Technology, Danvers, MA, USA, 2108S) in TBST overnight at +4 °C.

Techniques: Sequencing

Journal: Cells

Article Title: Na,K-ATPase Acts as a Beta-Amyloid Receptor Triggering Src Kinase Activation

doi: 10.3390/cells11172753

Figure Lengend Snippet: Aβ 42 alters cellular redox parameters and does not activate Src kinase in hypoxic conditions. ( A , B ) The effect of Aβ 42 on the levels of reactive oxygen species (ROS). ( C , D ) The oxidized glutathione/reduced glutathione (GSSG/GSH) ratio. ( E , F ) Reduced glutathione (GSH), and ( G , H ) Ca 2+ levels in the SH-SY5Y human neuroblastoma cells. The cells were harvested, stained with fluorescent probes: Dyhydrorhodamine 123 for ROS measurements, ThiolTracker Violet for GSH measurements, and Fluo-4 for Ca 2+ levels measurements and incubated with 100 nM Aβ 42 for 10 min ( A , C , E , G ) or for 30 min ( Β , D , F , H ). The GSSG/GSH ratio was determined with Grx1-roGFP genetically encoded ratiometric sensor. The change in the GSSG/GSH ratio was determined by calculating the ratio of the fluorescence intensity values at a wavelength of 535 nm, obtained with excitation at the wavelengths of 488 and 400 nm. All parameters were normalized for control. ( I , J ) The ratio of phospho(Tyr-416)-Src to total Src in SH-SY5Y neuroblastoma cells incubated with 100 nM of Aβ 42 for 30 min under hypoxic conditions (1% O 2 ) and standard conditions (20% O 2 ) determined with Western blot. ( I ) The representative blot, and ( J ) the corresponding p-Src/Src ratio are presented. Mean values ± SD from at least three independent experiments are shown. *— p <0.05, **— p <0.01 compared to the control, ns—nonsignificant.

Article Snippet: The membrane was blocked in 5% nonfat milk in TBST (50 mM Tis-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20), and incubated with primary rabbit antibodies to phospho (Tyr 416)-Src kinase (p-Src; Cell Signaling Technology, Danvers, MA, USA, 6943S) or total Src kinase (Cell Signaling Technology, Danvers, MA, USA, 2108S) in TBST overnight at +4 °C.

Techniques: Staining, Incubation, Fluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Pannexin-1 Activation by Phosphorylation Is Crucial for Platelet Aggregation and Thrombus Formation

doi: 10.3390/ijms23095059

Figure Lengend Snippet: PANX1 represents a platelet ATP channel and is phosphorylated after CRP and PAR4 peptide stimulation of human platelets via Src activation: ( A , B ) Isolated platelets were pre-incubated with the PANX1 inhibitor Prb and ATP release was measured in resting platelets and after activation with ( A ) 1 µg/mL CRP ( n = 6) via ATP ELISA or ( B ) 1 and 5 µg/mL CRP ( n = 4–6) and 70 and 200 µM PAR4 ( n = 4–6) via a luciferin/luciferase bioluminescent assay (aggregometer). ( C – F ) Immunoblotting was performed using isolated platelets activated with indicated concentrations of CRP and PAR4. ( C ) Phosphorylation of PANX1 at Tyr 198 was quantified (n= 8) and ( D ) analyzed after pre-incubation of platelets with the SFK inhibiting compound PP2 or PP3 as a negative control ( n = 6). ( E ) Phosphorylation of Src at Tyr 416 was analyzed in platelets isolated from Panx1 fl/fl Pf4-Cre + (KO) and Panx1 fl/fl Pf4-Cre − (WT) mice following CRP stimulation ( n = 4). ( F ) Phosphorylation of PANX1 at Tyr 198 was analyzed in platelets from WT and Gp6 −/− mice ( n = 4). Statistical analysis was performed using a two-way ANOVA ( D , E compared to resting; A – C , F , G between groups) followed by a Sidak’s multiple comparisons post-hoc test. Bar graphs indicate mean values ± SEM, * p < 0.05: ** p < 0.01 and *** p < 0.001. Rest = resting, CRP = Collagen-related peptide, PAR4 = Protease-activated receptor 4 peptide, Prb = probenecid, SFK = src family kinase, WT = wild type, KO = knockout.

Article Snippet: Antibodies against phosphoPANX1 Tyr 198 (Merck, Darmstadt, Germany; cat no ABN1681); phosphoPANX1Tyr 308 (Merck, Darmstadt, Germany; cat no ABN1680); PANX1 (Cell Signaling, Cambridge, UK; cat no 91137S); phosphoSrc Tyr 416 (Cell Signaling, Cambridge, UK; cat no 2101); Src (Cell Signaling, Cambridge, UK; cat no 2108); phosphoAkt Ser 473 (Cell Signaling, Cambridge, UK; cat no 9271S); Akt (Cell Signaling, Cambridge, UK; cat no 9272S); GAPDH (Cell Signaling, Cambridge, UK; cat no 2118S); β-Actin (Cell Signaling, Cambridge, UK; cat no 4967) and fibrinogen (Dako, Jena, Germany; cat no A0080) were used for immunoblotting.

Techniques: Activation Assay, Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Luciferase, Western Blot, Negative Control, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation

doi: 10.3390/ijms23094996

Figure Lengend Snippet: Effect of 3α-THP on c-Src activation in U251 GB cells in time-course experiments. The phosphorylation of c-Src (Y416) was determined under the treatments of 3α-THP 100 nM or Vehicle (V, EtOH 0.01%) at ( a ) 5 min; ( b ) 10 min; ( c ) 15 min; ( d ) 30 min by Western blot. The upper panel of each section shows a representative Western blot experiment of p-c-Src, total c-Src, and α-Tubulin as a loading control. Each lower panel presents the densitometric analysis of p-c-Src/c-Src. Each column represents the mean ± SEM., n = 3; * p < 0.05 3α-THP vs. V.

Article Snippet: When c-Src activation was evaluated, the membranes were first incubated with the primary antibodies for the phosphorylated or total forms of c-Src: phospho c-Src Tyr-416 (1:1000; Ref. 2101; Cell Signaling, Massachusetts, MA, USA), and c-Src (1:1000; Ref. 2108, Cell Signaling, Massachusetts, MA, USA).

Techniques: Activation Assay, Western Blot