Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Spinal injection of TNF-α-activated astrocytes induces mechanical allodynia via Cx43-mediated CXCL1 release. (A) Intrathecal injection of TNF-α-activated astrocytes elicited persistent mechanical allodynia for >48 h. Note this allodynia is reduced by pretreatment of astrocytes with Cx43 small interfering RNA (1 µg/ml, 18 h). *P < 0.05, compared with TNF-α or TNF-α + non-targeting control small interfering RNA treated group; n = 6 mice/group. (B) ELISA analysis shows increased CXCL1 release in the CSF at 3 h after the intrathecal injection of TNF-α-activated astrocytes. *P < 0.05, compared with vehcile group; #P < 0.05, compared with non-activated astrocytes; n = 4 mice/group. (C) Intrathecal injection of a CXCL1 neutralizing antibody (4 µg) transiently and partially reversed mechanical allodynia, induced by TNF-α-treated astrocytes. *P < 0.05, compared with control IgG group; n = 6 mice/group. (D) Intrathecal injection of the CXCR2 antagonist SB225002 (20 µg = 57 nmol) transiently and partially reversed mechanical allodynia, induced by TNF-α-activated astrocytes. *P < 0.05, compared with vehicle (PBS); n = 5–6 mice/group. All data are mean ± SEM. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Injection, Small Interfering RNA, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: CXCL1, upregulated in spinal cord astrocytes after nerve injury, enhances excitatory synaptic transmission in spinal cord neurons and maintains neuropathic pain via CXCR2. (A) Western blotting shows CXCL1 upregulation in the spinal cord dorsal horn 21 days after CCI. Right, quantification of Cx43 levels in the dorsal horn. The western blot results are presented as a fold of sham control. *P < 0.05, compared to sham control, Student’s t-test, n = 4 mice/group. (B) Intrathecal injection of SB 225002 (20 µg), 21 days after CCI, reduced CCI-induced mechanical allodynia in the late phase. *P < 0.05, compared with vehicle (saline), Student’s t-test, n = 6 mice/group. (C) Double immunostaining of CXCL1 and GFAP in the dorsal horn 21 days after CCI. Note CXCL1 is primarily colocalized with GFAP. Arrows indicate doubled-labelled cells. Scale bar = 50 µm. (D and E) CXCL1 superfusion (100 ng/ml) increases spontaneous EPSC frequency (revealed by patch clamp recordings) in lamina IIo neurons of spinal cord slices. (E) Spontaneous EPSC frequency. *P < 0.05, Student’s t-test, n = 5 neurons/group. (F and G) CCI (21 d) increases spontaneous EPSC frequency, which is reversed by the CXCR2 antagonist SB225002 (1 µM). *P < 0.05, Student’s t-test, n = 5 neurons/group.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Transmission Assay, Western Blot, Control, Injection, Saline, Double Immunostaining, Patch Clamp

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Schematic of working hypothesis for astrocytic Cx43-mediated late-phase neuropathic pain. CCI induces a persistent upregulation of Cx43 in spinal cord astrocytes. Cx43 expression and activity is also upregulated by TNF-α, secreted from microglia. Upregulation of Cx43 hemichannel activities results in CXCL1 release. Astrocytic CXCL1 secretion activates CXCR2 on neurons (central terminals of primary sensory neurons and spinal cord neurons), leading to enhanced excitatory synaptic transmission in nociceptive neurons (e.g. lamina IIo excitatory interneurons) and sustained neuropathic pain in the late-phase. Additionally, CXCL1 can also be secreted from intact or injured primary afferents in the spinal cord especially in the early phase of CCI.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Expressing, Activity Assay, Transmission Assay

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) Cell surface N-glycoproteins that are significantly altered in cell surface abundance upon exposure to SV40 are visualized with a network view. The glycoproteins whose abundance was either increased (yellow) or decreased (blue) during exposure to SV40 (and which either did not change during exposure to VSV, or changed in the opposite direction compared to SV40) are depicted as nodes in the network. The different shades represent different degrees of relative abandunce (log2 values). The remaining nodes in the network are the hits from the RNAi screen (see ), which either increased (green) or decreased (red) SV40 infection upon siRNA knockdown. For the common hits in the CSC and RNAi screens, the node border represents the RNAi phenotype (ITGA6, ITGB6 and CD47 were CSC-hits but gave no RNAi phenotype when tested). The grey connecting lines between nodes illustrate protein interactions, which were assessed using the STRING database with a combined score of at least 0.9 , and were visualized using Cytoscape ( www.cytoscape.org ) and the Cerebral plugin .

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Infection, Knockdown

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) A targeted siRNA screen reveals several structural and signaling components of cell adhesion to regulate the SV40 infectious route. A set of four siRNAs against 263 genes was applied in A431 human epithelial cells and virus infection was assessed by the presence of nuclear large T-antigen. Low-resolution imaging and image processing with the CellProfiler analysis software were subsequently performed. A Support Vector Machine (SVM)-based classification method was then used to determine percentage of infection upon siRNA treatment. The table shows the genes that reduced (red shades) or enhanced (green shades) SV40 infection with different strength when knocked down. The values in the boxes represent the number of different siRNAs that gave a similar phenotype. (B) Epistasis analysis between Cav1, GRAF1, and Ezrin. A431 cells were treated with siRNA against each one of these genes or combinations of two. Two or three siRNAs were used per gene. Cells were subsequently treated with SV40 and infection levels were assessed by the presence of nuclear T-antigen. The graph shows values pooled from the individual infection indices. p-values: 1.3×10 −4 (Ezrin-siRNA, Cav1-siRNA), 0.39 (Ezrin-siRNA, GRAF1-siRNA). (C) Blocking integrin α2 function with an antibody reduces SV40 infection, similar to siRNA-mediated knock down. A431 cells were pre-incubated with 0.02 µg/µL of blocking antibody 20 min prior to infection (p-values 1×10 −4– 7×10 −4 ). (D) siRNA against integrins α2 and β1 reduces binding of SV40 at the surface of A431 cells. Binding was performed at cold for 2 h and binding capacity was determined by immunoblotting for the presence of the major capsid protein VP1 in cell extracts. Signal intensity was quantified by the ImageJ software and standard deviation corresponds to two independent experiments. (E) SV40 binds onto the surface of various cell lines with different intensity; GM1-deficient cells retain the ability to bind SV40. Quantification of signal intensity from two independent experiments was performed as in (D). (F) SV40-like particles (VLPs) can bind cells that lack its native receptor GM1 in a dose-dependent manner. (G) SV40 can bind cells that lack its native receptor GM1 via integrins. GM95 cells were treated with siRNA against integrin α2 and SV40 binding was determined by the abundance of VP1 protein, as described in (D). (H) Integrins can serve as binding sites for SV40. Integrin α2β1 was immunoprecipitated from A431 cells pretreated for 2 h with SV40 in the cold, and the VP1 protein was detected in the immunocomplex by immunoblotting (red box). Transferrin receptor was used as a negative control.

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Virus, Infection, Imaging, Software, Plasmid Preparation, Blocking Assay, Knockdown, Incubation, Binding Assay, Western Blot, Standard Deviation, Immunoprecipitation, Negative Control

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) AKT is activated in a wortmannin-sensitive manner in A431 cells following SV40 treatment. A431 cells were treated with wortmannin for 1 h prior to SV40 treatment. Phosphorylated AKT at Ser473 was tested by immunoblotting. (B, C) Phosphorylated AKT is recruited to the plasma membrane of A431 cells in a cholesterol-dependent manner. p-AKT (Ser473) was detected by immunofluorescence in fixed cells (B) or immunoblotting (C), before or 30 min after SV40 treatment. Cholesterol was extracted from the plasma membrane using methyl-β-cyclodextrin (mβCD) for 45 min. Cholesterol re-administration was succeeded with cholesterol coupled to mβCD for 3 h. Scale bars in (B) represent 15 µm. (D) Incubating A431 cells with an antibody against integrin α2β1 results in activation of AKT signaling. Incubation was carried out for 15 min before the addition of SV40 for another 10 min. Cholesterol extraction prior to antibody application was performed as described in (B). (E, F) siRNA-mediated knockdown of human Integrin Linked Kinase (ILK) (E) and integrins α2 and β1 (F) leads to defective activation of AKT in A431 cells, 10 min following SV40 treatment. (G) AKT is phosphorylated in GM95 cells following SV40 treatment. Quantification of all western blots was done with the ImageJ software; values represent averages of two independent experiments ± standard deviation.

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Western Blot, Clinical Proteomics, Membrane, Immunofluorescence, Activation Assay, Incubation, Extraction, Knockdown, Software, Standard Deviation

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) Cortical actin alterations occur in A431 cells following incubation with SV40 for 10 min, as visualized with phalloidin staining in fixed cells. (B) Phosphorylated ERM proteins become inactive shortly after SV40 treatment. A431 cells were treated with SV40 for the indicated time points, fixed and immunostained with an antibody against phosphorylated T567 of ERM proteins. p-ERM disappears as soon as 5 min after incubation with SV40 and it resumes after 1 h. (C) A phosphomimetic mutant form of Ezrin, T567D, fails to fall off the membrane following SV40 treatment. A431 cells were transfected with EzrinT567D-GFP, treated with SV40 for 15 min, and subsequently fixed and tested for the GFP signal localization. (D) Expression of both a phosphomimetic and an inactive Ezrin mutant form negatively correlates with SV40 infection. Large populations of A431 cells were transfected with EzrinT567D, EzrinT567A or wild-type Ezrin GFP constructs and subjected to SV40. Cells that were both transfected (GFP signal) and infected (T-antigen signal) were scored and compared with the expected number emerging from a random occurrence of the two signals. Negative log2 ratio values represent an anti-correlation, which demonstrates inhibition of infection by the transfected construct (p-value 6.8×10 −3 and 0.05 for the T567D and T567A scores, respectively). (E, F) Inhibition of PDK1 function leads to increased levels of p-ERM. The PDK1 inhibitor was applied onto A431 cells for 1.5 h before the addition of SV40 for another 15 min. Levels of p-ERM were assessed using either immunofluorescence in fixed cells (E) or immunoblotting (F). Quantification of two different blots was performed using the ImageJ software. (G) Inhibition of PDK1 function blocks increased levels of infection as caused by Ezrin knockdown. A431 cells were subjected to siRNA against Ezrin or control siRNA, and subsequently treated with the PDK1 inhibitor 1.5 h prior to SV40 infection, or left untreated. Percentage of infection was calculated from a large number of cells. p-values: 1×10 −4 (PDK1 inhibitor), 5.4×10 −3 (Ezrin siRNA), 5.8×10 −4 (Ezrin siRNA, PDK1 inhibitor).

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Incubation, Staining, Mutagenesis, Membrane, Transfection, Expressing, Infection, Construct, Inhibition, Immunofluorescence, Western Blot, Software, Knockdown, Control

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) Constitutively active RhoA leads to increased levels of phosphorylated Ezrin that persist upon SV40 treatment. Inactive RhoA abrogates basal levels of p-ERM. A431 cells were transfected with RhoA-G14V-GFP or RhoA-T19N-GFP before SV40 was applied for 15 min and fixed cells were stained with a p-ERM antibody. (B) Expression of an inactive RhoA mutant form positively correlates with SV40 infection. Large populations of A431 cells were transfected with RhoA-G14V, RhoA-T19N and wild-type RhoA GFP constructs and subjected to SV40. Cells that were both transfected (GFP signal) and infected (T-antigen signal) were scored and compared with the expected number emerging from a random occurrence of the two signals. Positive log2 ratio values represent a positive correlation, demonstrating a stimulation of infection, whereas negative values denote anti-correlation, demonstrating an inhibition of infection (p-value 4.2×10 −4 ). (C) Inhibition of GRAF1 function leads to increased levels of p-ERM. siRNA was applied onto A431 cells followed by addition of SV40 for 15 min. Levels of p-ERM were assessed using immunofluorescence in fixed cells. (D) RhoA is inactivated 10 min after SV40 treatment. Inhibition of PI3K and PDK1 with wortmannin or the PDK1 inhibitor, respectively, abolished the SV40-induced reduction in RhoA activity. Cells that had undergone the indicated treatment were subjected to RhoA-GTP immunoprecipitation, which was subsequently detected with a RhoA antibody using immunoblotting. The graph shows the quantification of the RhoA-GTP signal in SV40 exposed cells, as expressed in % reduction compared to the non-treated cells, and normalized against total RhoA and tubulin (quantification based on two different experiments). (E) PDK1 and GRAF1 act both upstream of RhoA to signal to ERM proteins. A431 cells were subjected to the following conditions before being scored for the presence or absence of p-ERM signal using immunofluorescence: transfection with RhoA-WT-GFP, RhoA-G14V-GFP or RhoA-T19N-GFP constucts, incubation with the PDK1 inhibitor for 1.5 h, siRNA treatment against GRAF1, or a combination of RhoA-T19N-GFP expression and the PDK1 inhibitor or GRAF1 siRNA. Acquired confocal images were processed with ImageJ to quantify the number of p-ERM-expressing cells. Inhibition of PDK1 function or silencing of GRAF1 led to partial or no restoration of the fraction of p-ERM-positive RhoA-T19N-expressing cells (asterisks), respectively. Values shown are the average of 2–4 independent experiments ± standard deviation. (F) Representative image used to extract the values shown in (E). The white line outlines manually segmented cells, whereas green and red depict RhoA-T19N-GFP transfected cells and p-ERM-positive cells, respectively. Dapi-stained nuclei are shown in blue.

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques: Transfection, Staining, Expressing, Mutagenesis, Infection, Construct, Inhibition, Immunofluorescence, Activity Assay, Immunoprecipitation, Western Blot, Incubation, Standard Deviation

Journal: PLoS ONE

Article Title: Integrin-Mediated Signaling Induced by Simian Virus 40 Leads to Transient Uncoupling of Cortical Actin and the Plasma Membrane

doi: 10.1371/journal.pone.0055799

Figure Lengend Snippet: (A) The various players participating in the integrin-mediated signaling through host SV40 engagement. The respective time intervals during which these signaling events occur are also depicted.

Article Snippet: Antibodies were from the following sources: rabbit anti-p-ERM: Cell Signaling Technology; mouse anti-p-AKT (S473) and rabbit anti-p-AKT (S473): Upstate, clone 11E6 and Cell Signaling Technology, respectively; anti-p-p85 (Y458): Cell Signaling Technology; mouse integrin α2β1: abcam, clone P1E6; rabbit anti-SV40 VP1: abcam, ab53977; mouse anti-transferrin receptor: Zymed/Invitrogen, 13–6800; rabbit anti-beta tubulin: abcam, ab6046; mouse anti-RhoA: Santa Cruz, clone 26C4; Antisera against viral large T-antigen were provided by Dr. G. Brandner (University of Freiburg, Germany).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Immortalized mesenchymal stromal cells overexpressing alpha‐1 antitrypsin protect acinar cells from apoptotic and ferroptotic cell death

doi: 10.1111/jcmm.70093

Figure Lengend Snippet: Unconjugated and conjugated antibodies (with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)) employed for the current study.

Article Snippet: Rabbit Anti‐SV40 Large T Antigen , Transgenic levels of total SV40 Large T Antigen protein , IC, WB , Cell Signaling Technology , IC‐1:100, WB‐1:1000.

Techniques: Transgenic Assay, Bioprocessing

Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet: ( A, G ) Representative images of U251 glioblastoma cells ( A ) and N2A cells ( G ) immunolabelled for TDTOMATO (red) and F-actin (green) following transfection with plasmids encoding Myr-TDTOMATO, DCC:TDTOMATO or DCC kanga :TDTOMATO demonstrating the presence of actin-rich regions resembling filopodia (yellow arrows), lamellipodia (yellow arrowheads) and membrane ruffles (yellow asterisks) with/without stimulation with recombinant mouse NTN1 protein. ( B ) Schema of predicted structure of proteins on the cell membrane encoded by the plasmids expressed in cells from ( A ) and ( G ). ( C, H ) Outline of cell perimeter generated from images in ( A ) and ( G ), respectively. ( D–F, I ) Quantification of the area, perimeter and circularity of cells represented in ( A ) and ( G ). Graphs represent mean ± SEM. Statistics by Kruskal–Wallis test for multiple comparisons. n.s: not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See related and . Figure 7—source data 1. U251 or N2A cell area, perimeter and circularity following overexpression of DCC:TDTOMATO or myr-TDTOMATO.

Article Snippet: GADPH was used as a loading control and detected using rabbit monoclonal anti-GADPH antibodies (1:2000, 2118, Cell Signaling Technology for COS-7; 1:1000, IMG-5143A, IMGENEX for N2A and U251).

Techniques: Transfection, Recombinant, Generated, Over Expression

Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet:

Article Snippet: GADPH was used as a loading control and detected using rabbit monoclonal anti-GADPH antibodies (1:2000, 2118, Cell Signaling Technology for COS-7; 1:1000, IMG-5143A, IMGENEX for N2A and U251).

Techniques: Droplet Countercurrent Chromatography, Western Blot, Immunofluorescence, Recombinant, shRNA, CRISPR, In Situ Hybridization, Sequencing, Mutagenesis, Generated, Imaging, Software

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Anti-apoptotic property of full-length ADAM15. A, ADAM15-, ADAM15Δcyto-, and vector-transfected cells were treated with 20 μm camptothecin and caspase-3/7 activity as a marker for apoptosis was measured for 0–10 h. B, the ATP content, which correlates with cell viability, was determined after exposure of the cells for 18 h using increasing camptothecin concentrations. Both assays show that the removal of the cytoplasmic tail of ADAM15 resulted in a loss of the anti-apoptotic and cell protective capacity compared with chondrocytes transfected with full-length ADAM15. *, p < 0.0004, when comparing the caspase activity or viability of the ADAM15- versus vector-transfected cells.

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Plasmid Preparation, Transfection, Activity Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Enhanced phosphorylation of FAK in ADAM15-transfected chondrocytes after apoptosis induction by camptothecin. A, immunoblots of cell lysates of vector-transfected (−) and ADAM15-transfected cells (+) treated with 2 μm camptothecin for up to 60 min, showing a stronger phosphorylation at Tyr-576, Tyr-861, and Tyr-397 of FAK in the ADAM15-expressing cells in comparison with vector-transfected cells. B, T/C28a4 cells transfected with ADAM15 lacking the cytoplasmic tail (ΔC), however, did not display an activation of FAK. Blots were controlled for ADAM15 and/or ADAM15ΔC expression, and loading was monitored using anti tubulin antibodies. The immunoblots are representative of at least five repeated experiments.

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Phospho-proteomics, Transfection, Western Blot, Plasmid Preparation, Expressing, Comparison, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Generation of recombinant ADAM15 and FAK proteins for protein binding assays. A, upper left panel: Coomassie stained SDS-PAGE of the purified Gst-tagged FAK fragments and the Gst- and Myc-tagged cytoplasmic domain of ADAM15 (cyto) and upper right panel: the detection of these proteins with anti-Gst antibodies by Western blotting. A, lower panels: SDS-PAGE of the cytoplasmic domain of ADAM15 with the Gst tag cleaved off by PreScission Protease, followed by immunodetection using anti-Myc and anti-ADAM15 antibodies. B, pulldown assays: cell lysates spiked with Myc-tagged ADAM15 were co-incubated with either of the three different FAK proteins. Upon immunoprecipitation (IP) using anti-Gst-Sepharose and subsequent immunoblotting (IB), bound ADAM15 was detected by anti-Myc antibodies. Blots were stripped, and the Gst-tagged FAK proteins were verified by anti-Gst antibodies, thereby demonstrating the interaction of ADAM15 with the C-terminal FAK-region (amino acids 707–913).

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Recombinant, Protein Binding, Staining, SDS Page, Purification, Western Blot, Immunodetection, Incubation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Interaction of ADAM15 with FAK. A, the domain structure of FAK: the FERM, the catalytic domain, and the C-terminal domain with the FAT domain. Black bars, two proline-rich regions (PXXP motifs) are binding sites for SH2/SH3 domain containing proteins. B and C, mammalian two-hybrid assay. The distinct FAK domains, cloned as segregated fragments into the pCMV-BD bait vector, were co-transfected with the cytoplasmic domain of ADAM15 in pCMV-AD prey vector and a firefly luciferase reporter into HEK293 cells. B, an exclusive interaction of ADAM15 with a C-terminal region of FAK (amino acids 707–913), but not with the FERM, the kinase, or the FAT domain is demonstrated. Mutation of Tyr-861 into Phe-861 in the interaction domain of FAK had no influence on ADAM15 binding. C, left panel, further fine-mapping of the Fak domain 707–913 revealed fragment 730–790 (underlined) as the smallest region with binding capacity equivalent to 707–913. Shortening of 10 amino acids either at the N or C terminus abrogates the binding to ADAM15 by ∼70%. Shown is a representative experiment. C, right panel, depicts the mean (± S.D.) of five repeated assays of ADAM15 binding with each distinct FAK fragment. Measured luciferase values were normalized to the values obtained from a co-transfected Renilla luciferase control plasmid.

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Binding Assay, Two Hybrid Assay, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Mutagenesis, Control

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Co-localization of ADAM15 and FAK. ADAM15 transfected chondrocytic T/C28a4 cells (A) and human osteoarthritic chondrocytes (B) were grown on collagen type II and double-stained with goat anti-ADAM15 and mouse anti-FAK antibodies visualized with Alexa Fluor 488 (green) and 594 (red) conjugated secondary antibodies using confocal laser scanning microscopy (40× objective). C, shown is a magnification of the white boxed area in B with co-localized spots marked by white arrows. The merged images clearly reveal a co-localization of ADAM15 and FAK in focal contacts.

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Transfection, Staining, Confocal Laser Scanning Microscopy

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Stronger phosphorylation of Src kinase in ADAM15-transfected cells is dependent on direct binding to FAK. A, upon stimulation with camptothecin for 60 min, an enhanced phosphorylation of Tyr-416 Src was detected in whole cell lysates of ADAM15-transfected cells (+) as compared with those derived from respective vector control cells (−) by immunoblotting. B, to analyze whether the stronger phosphorylation of Src results from an interaction with ADAM15 or with FAK, co-immunoprecipitations (IP) were performed by using as the precipitating antibody either 1) anti-ADAM15 or 2) anti FAK. C, lysates of ADAM15 (+) and vector control cells (−) upon camptothecin exposure were immunoprecipitated with either anti-ADAM15 1) or anti-FAK antibodies 2) and immunoblotted using anti Tyr-416 Src antibodies. A stronger phosphorylation of Tyr-416 Src was observed in the ADAM15-transfected cells compared with the vector control cells, independent of the antibody used for the precipitation. Blots were stripped, and precipitated Src was visualized using anti-Src antibodies. Src was pulled down in ADAM15 precipitated lysates only (vector-transfected cells, negative). In all anti-FAK antibody-precipitated lysates, equal amounts of Src protein were demonstrated. Blots were restripped and controlled for precipitated ADAM15 1) or FAK 2) using respective antibodies. D, mammalian two-hybrid was used to analyze the binding partner of Src. The N-terminal SH2/SH3 domain as well as full-length Src were cloned into the prey vector and distinct FAK domains into bait vector. Bait and prey were co-transfected with the firefly luciferase reporter and a Renilla luciferase control plasmid into HEK cells. A strong binding of the SH2/SH3 domain as well as full-length Src to the C-terminal region of FAK (707–913) was measured, whereas the FERM, the catalytic, and the FAT domain did not bind to Src. Mutation of Tyr-861 into Phe-861 of FAK did not affect Src interaction. However, no direct binding of ADAM15 to SH2/SH3 Src or full-length Src could be detected by contrast to the already proven interaction with the C-terminal FAK domain (707–1052, Fig. 4) that served as a positive control in this experiment.

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Phospho-proteomics, Transfection, Binding Assay, Derivative Assay, Plasmid Preparation, Control, Western Blot, Immunoprecipitation, Clone Assay, Luciferase, Mutagenesis, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Signaling of ADAM15 expressed as a IL-2 receptor-α/cytoADAM15 chimera. The T/C28a4 chondrocyte cell line was stably transfected with a chimeric construct composed of the extracellular IL-2 receptor-α (CD25) fused to the cytoplasmic tail of ADAM15, and the chimeric protein was detected by immunoblotting using either anti-CD25 or anti-ADAM15 antibodies at ∼60 kDa (A, left panels). A, right panel, shows co-immunoprecipitation of FAK only in cell lysates of cells transfected with the chimeric IL2Rα/cytoADAM15 construct (Ch) as compared with vector transfected cells (−) using anti-CD25 antibodies. B, immunoprecipitation (IP) using anti-FAK antibodies show co-precipitated Src in both vector and chimera-transfected cells, but an enhanced phosphorylation of Tyr-416 Src is noted exclusively in the IL2Rα/cytoADAM15 cells following stimulation with IL-2. C, immunoblots (IB) of lysates derived from IL-2 stimulated cells exhibit a stronger phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 in IL2Rα/cytoADAM15 cells. Equal loading was controlled by reprobing with anti-FAK antibodies.

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Stable Transfection, Transfection, Construct, Western Blot, Immunoprecipitation, Plasmid Preparation, Phospho-proteomics, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Inhibition of FAK and Src signaling by FAK 14 inhibitior and PP2 in the presence of ADAM15. Vector- (black bars) and ADAM15-transfected cells (open bars) were treated with 0.25 μm and 0.5 μm FAK 14 inhibitor (A) and 5 nm and 10 nm PP2 (B) for the time points shown, and caspase-3/7 activity was determined. A significantly increased caspase activity was detected in the control cells in contrast to ADAM15-transfected cells that exhibited higher apoptosis resistance to both inhibitors. Incubation with DMEM alone also displayed a significantly higher caspase activity in vector control cells. C and D, ADAM15 transfected cells were silenced using siRNA I and II for 40 h and treatment with 0.25 μm FAK 14 inhibitor (C) or 5 nm PP2 (D) resulted in a significantly higher caspase activity as compared with cells silenced with a nonfunctional siRNA (N) or with transfection agent alone (0). Incubation with DMEM alone served as a control. Shown are representative results of least five repeated experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Inhibition, Plasmid Preparation, Transfection, Activity Assay, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Enhanced phosphorylation of FAK and Src in osteoarthritic chondrocytes after apoptosis induction by camptothecin. Immunoblots of cell lysates of OA chondrocytes that were silenced using ADAM15-specific siRNAs I and II and a nonfunctional siRNA (N) or transfection agent alone (0) and treated with camptothecin, showing marked induction of phosphorylation of FAK at Tyr-576, Tyr-861, and Tyr-397 and Src at Tyr-416 by camptothecin after 15 and 30 min (N and 0), which is strongly reduced after silencing of ADAM15 (I and II). Shown are representative results of an analysis performed on primary chondrocytes derived from five OA cartilage samples.

Article Snippet: Mouse and goat anti-ADAM15 antibodies were from R&D Systems (recognizing the prodomain between amino acids 1–200 ( 14 ) catalog no. MAB935, AF935).

Techniques: Phospho-proteomics, Western Blot, Transfection, Derivative Assay