Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: F1000Research

Article Title: Murine Tim-1 is excluded from the immunological synapse

doi: 10.12688/f1000research.1-10.v2

Figure Lengend Snippet: ( A ) Jurkat T cells transfected with empty vector (EV), Flag-Tim-1, or Flag-Tim-1 QGQ were stimulated with anti-TCR and anti-CD28 antibodies for the indicated times. Lysates were analyzed by SDS-PAGE and western blotting for pY (4G10), pSrc (Y416; analogous to Y394 in Lck), and β-actin. ( B ) Jurkat T cells were stimulated with anti-CD3 mAb for the indicated times. CD3 expression was measured with flow cytometry and mean fluorescence intensity (MFI) was determined in FloJo. Dynasore (DS, 80 µM) was used to prevent clathrin-mediated endocytosis after TCR/CD3 crosslinking.

Article Snippet: The following antibodies were used: pSrc Y416 and pZAP-70 Y319 (Cell Signaling), PKC-θ (C-18, Santa Cruz), CD43 (clone S7, BD Pharmingen), M2-Cy3 (Sigma Aldrich), EEA1 (BD Transduction), M2 anti-flag (Sigma Aldrich), anti-human CD3 (Becton Dickinson), mouse CD3 and CD28 (BD Pharmingen), human CD28 (Life Technologies), Tim-1 Fc (eBiosciences), anti-TCR antibody C305 (Harlan), anti-Tim-1 antibodies (3B3 and 5F12), anti-Tim-4 antibodies (3A1, 3H11 and 5G3).

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot, Expressing, Flow Cytometry, Fluorescence

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 1. ICAM-1 cross-linking in- duced time-dependent activation of SRC tyrosine kinases. TNF--pre- treated ECs were incubated for 30 min with 10 g/ml mouse anti-human ICAM-1 antibody and washed. A cross-linking sec- ondary antibody was added for 0–15 min, and the activity of SRC was evaluated by an in vitro kinase assay using Sam68- (331–443) as a substrate after immuno- precipitating SRC. Tyrosine phosphoryla- tion of Sam68-(331–443) was detected by immunoblot using an anti-phosphoty- rosine antibody as described under “Ex- perimental Procedures.” A, representa- tive immunoblot showing activation of SRC tyrosine kinases in response to ICAM-1 cross-linking. The amount of im- munoprecipitated SRC was also deter- mined for each sample. Lanes 1–5, SRC activity in ECs prior to (lane 1) or 0.25–15 min after ICAM-1 cross-linking (lanes 2–5). B, densitometric analysis of immu- noblots. The activity of SRC tyrosine ki- nases was normalized by the amount of immunoprecipitated SRC. Data are pre- sented as fold changes over the non-cross- linked controls and expressed as means S.E. (n 4). *, p 0.05 when compared with controls.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Activity Assay, In Vitro, Kinase Assay, Western Blot, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 2. Activation of SRC tyrosine ki- nases was inhibited by allopurinol, a xanthine oxidase inhibitor (A), as well as Me2SO, a hydroxyl radical scav- enger, and deferoxamine, an iron che- lator (B). ECs were treated with 10 g/ml anti-ICAM-1 along with 0.3 mg/ml allo- purinol, 1% Me2SO, or 1.5 mM deferoxa- mine, or their respective control vehicle for 30 min and washed. A cross-linking sec- ondary antibody was added for 0–6 min, and SRC activity was evaluated as de- scribed under “Experimental Procedures.” Open bars, no cross-linking; closed bars, cross-linking for 6 min. Data are expressed as fold changes from the non-cross-linked controls in vehicle-pretreated samples and presented as mean S.E. (n 4). *, p 0.05 when compared with the non-cross- linked controls; #, p 0.05 when compared with the vehicle-pretreated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Control, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 3. Modulation of SRC activity by PAO, a tyrosine phosphatase in- hibitor. ECs were treated with 10 g/ml anti-ICAM-1 along with control vehicle or 20 M PAO for 30 min and washed. A cross-linking secondary antibody was added for 0–6 min, and SRC activity was evaluated as described under “Experi- mental Procedures.” Data are expressed as fold changes from the non-cross-linked controls in vehicle-pretreated samples, and presented as mean S.E. (n 4). *, p 0.05 when compared with the non- cross-linked controls; #, p 0.05 when compared with the vehicle-pretreated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Control

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 4. Activation of SRC tyrosine kinases required SHP-2. ECs were treated with 10 nM control or SHP-2 an- tisense oligonucleotides as described un- der “Experimental Procedures.” A, the ef- fect of SHP-2 antisense on the protein expression of SHP-2 or SHP-1 in ECs as examined by immunoblot. B, the effect of SHP-2 antisense on SRC activity before or after ICAM-1 cross-linking for 6 min. Data are expressed as fold changes from the non-cross-linked controls and pre- sented as means S.E. (n 8). *, p 0.05 when compared with the non-cross- linked controls; #, p 0.05 when com- pared with the control antisense-treated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Control, Expressing, Western Blot, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 5. Immunoprecipitated SHP-2 from ECs can dephosphorylate the phospho-SRC peptide at residue Tyr-530. SHP-2 was immunoprecipitated (IP) from ECs, and dephosphorylation of the phospho-SRC peptide at residue Tyr-530 was examined as described under “Experimental Procedures.” A, examples of two independent samples showing that immunoprecipitated SHP-2 can decrease the phosphorylation levels of the phospho-SRC peptide. Top gel, SHP-2 was specifically immunoprecipitated using a SHP-2 antibody. Bottom gel, incubation with the immunoprecipitated SHP-2 resulted in a decrease in the phosphorylation levels of the phospho-SRC peptide. B, densitometric analysis of the decrease in the phosphorylation levels of the phospho-SRC peptide as shown in A. Data are expressed relative to the control samples and presented as means S.E. (n 5). *, p 0.05 when compared with control samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Residue, De-Phosphorylation Assay, Phospho-proteomics, Incubation, Control

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 6. Activation of p38 MAPK in- duced by ICAM-1 cross-linking was inhibited by PP2, an inhibitor of SRC tyrosine kinases. ECs were incubated with 10 g/ml anti-ICAM-1 antibody along with vehicle or 20 M PP2 for 30 min and washed. The cells were either left untreated or treated with cross-linking secondary an- tibody for 6 min. The activity of p38 MAPK was evaluated by an in vitro kinase assay using ATF-2 as a substrate. A, activity of p38 MAPK as evaluated by phosphorylation of ATF-2. As a loading control, the amount of ezrin in the samples used for immunopre- cipitation was also examined. B, densitomet- ric analysis of ATF-2 phosphorylation as in A. Open bars, no cross-linking; closed bars, cross-linking ICAM-1 for 6 min. The data are expressed as fold changes from the non- cross-linked controls in vehicle-pretreated samples and are presented as means S.E. (n 6 or 7). *, p 0.05 when compared with the non-cross-linked controls.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Activity Assay, In Vitro, Kinase Assay, Phospho-proteomics, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Nicotine Induces Inhibitor of Differentiation-1 in a Src-dependent Pathway Promoting Metastasis and Chemoresistance in Pancreatic Adenocarcinoma 1

doi:

Figure Lengend Snippet: Depletion of Id1 abrogates chemoresistant phenotype and tumorigenic properties of pancreatic cancer cells. (A) Depletion of Src and Id1 using siRNA induces PARP cleavage in L3.6plGemRes-acquired.resistant pancreatic cancer cells exposed to gemcitabine therapy. (B) Depletion of Id1 expression in innate chemoresistant PANC-1 cells induces PARP cleavage and resensitizes cells to the cytotoxic effects of gemcitabine. (C) Tumor growth monitored by luciferase activity (photons per second) over approximately 4 weeks demonstrates increased tumor burden in mice treated with nicotine, whereas Id1 depletion resulted in inhibition of tumor growth regardless of nicotine exposure. (D) Graph showing the corresponding tumor volume (mm3) from each animal group corresponding to in vivo luciferase imaging of mice at termination of experiment (inset) demonstrating the promoting effects of nicotine and the suppressive effects of Id1 silencing on primary tumor growth. (E) Nicotine-induced pSrc and Id1 expression levels in primary tumors in vivo while having no effect on Id1 expression in tumors originating from cells with stably transfected shRNA silencing of Id1. (F) Ex vivo quantification of luciferase activity (photons per second) from livers demonstrates significantly higher liver metastasis in mice treated with nicotine while demonstrating no liver metastasis in mice with tumors derived from Id1-depleted cells. (G) H&E staining of livers (N) with metastatic pancreatic tumor demonstrates increased expression of Id1 in metastatic deposits (M).

Article Snippet: Monoclonal Id1 antibodies from BioCheck (Foster City, CA), monoclonal antibodies to c-Src (Upstate Biotechnology, Lake Placid, NY), p44/42 Erk MAPK and Akt (5G3) (Cell Signaling Technology, Beverly, MA), polyclonal α7 nAChR (Abcam, Cambridge, MA), phospho-Src (pSrc), phospho-Akt S473 , phospho-p44/42 Erk T202/Y204 , β-actin monoclonal antibody (Sigma, St Louis, MO), and PARP (Cell Signaling Technology) antibodies were used in Western blot analysis.

Techniques: Expressing, Luciferase, Activity Assay, Inhibition, In Vivo, Imaging, Stable Transfection, Transfection, shRNA, Ex Vivo, Derivative Assay, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Nicotine Induces Inhibitor of Differentiation-1 in a Src-dependent Pathway Promoting Metastasis and Chemoresistance in Pancreatic Adenocarcinoma 1

doi:

Figure Lengend Snippet: Id1 expression in resected human pancreatic adenocarcinoma. TMAs constructed from resected pancreatic ductal adenocarcinoma were stained for Id1, pSrc (activated form), and total Src using standard IHC techniques. A statistically significant correlation was observed between Id1 and tumor grade/differentiation and pSrc expression levels. No significant correlation was seen between total Src and Id1 expression.

Article Snippet: Monoclonal Id1 antibodies from BioCheck (Foster City, CA), monoclonal antibodies to c-Src (Upstate Biotechnology, Lake Placid, NY), p44/42 Erk MAPK and Akt (5G3) (Cell Signaling Technology, Beverly, MA), polyclonal α7 nAChR (Abcam, Cambridge, MA), phospho-Src (pSrc), phospho-Akt S473 , phospho-p44/42 Erk T202/Y204 , β-actin monoclonal antibody (Sigma, St Louis, MO), and PARP (Cell Signaling Technology) antibodies were used in Western blot analysis.

Techniques: Expressing, Construct, Staining