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: 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