Kinetworks&lt;sup&gt;TM&lt;/sup&gt; Protein Kinase Multiblot Analysis

Pelech, Steven; Sutter, Catherine; Zhang, Hong

doi:10.1385/1-59259-356-9:99

Cited by 35 publications

(38 citation statements)

References 3 publications

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“…1. Detailed information and protocols of the Kinetworks™ analysis have been published previously (25) and can also be found at the Kinexus Bioinformatics Corp. website (www.kinexus.ca).…”

Section: Methodsmentioning

confidence: 99%

B23/Nucleophosmin Serine 4 Phosphorylation Mediates Mitotic Functions of Polo-like Kinase 1

Zhang

Shi

Paddon

et al. 2004

Journal of Biological Chemistry

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“…1. Detailed information and protocols of the Kinetworks™ analysis have been published previously (25) and can also be found at the Kinexus Bioinformatics Corp. website (www.kinexus.ca).…”

Section: Methodsmentioning

confidence: 99%

B23/Nucleophosmin Serine 4 Phosphorylation Mediates Mitotic Functions of Polo-like Kinase 1

Zhang

Shi

Paddon

et al. 2004

Journal of Biological Chemistry

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“…Kinetworks Phospho-Site Screen 1.3 and KPSS-2.0 track 31 and 37 known phosphorylation sites, respectively in phosphoproteins with antibodies that recognise specific phosphorylated epitopes of the target proteins (Supplementary Tables S1 and S2). A total of 350 mg of whole cell lysates from H69 cells with or without HGF stimulation was used for each KPSS phosphoantibody array screen, which is an antibody-based method (Kinetworks) that relies on sodium dodecyl sulphate (SDS)-polyacrylamide minigel electrophoresis and multilane immunoblotters to permit the specific and quantitative detection of numerous protein kinases (PK) or other signal transduction proteins simultaneously (Pelech et al, 2003). Each blot was scanned densitometrically for quantitation, with each blot having its own unique Kinexus scan identification number.…”

Section: Phosphoantibody Array (Kinetworkt)mentioning

confidence: 99%

Downstream signalling and specific inhibition of c-MET/HGF pathway in small cell lung cancer: implications for tumour invasion

et al. 2007

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The c-MET receptor can be overexpressed, amplified, or mutated in solid tumours including small cell lung cancer (SCLC). In c-MET-overexpressing SCLC cell line NCI-H69, hepatocyte growth factor (HGF) dramatically induced c-MET phosphorylation at phosphoepitopes pY1230/1234/1235 (catalytic tyrosine kinase), pY1003 (juxtamembrane), and also of paxillin at pY31 (CRKL-binding site). We utilised a global proteomics phosphoantibody array approach to identify further c-MET/HGF signal transduction intermediates in SCLC. Strong HGF induction of specific phosphorylation sites in phosphoproteins involved in c-MET/HGF signal transduction was detected, namely adducin- α [S724], adducin- γ [S662], CREB [S133], ERK1 [T185/Y187], ERK1/2 [T202/Y204], ERK2 [T185/Y187], MAPKK (MEK) 1/2 [S221/S225], MAPKK (MEK) 3/6 [S189/S207], RB [S612], RB1 [S780], JNK [T183/Y185], STAT3 [S727], focal adhesion kinase (FAK) [Y576/S722/S910], p38 α -MAPK [T180/Y182], and AKT1[S473] and [T308]. Conversely, inhibition of phosphorylation by HGF in protein kinase C (PKC), protein kinase R (PKR), and also CDK1 was identified. Phosphoantibody-based immunohistochemical analysis of SCLC tumour tissue and microarray established the role of c-MET in SCLC biology. This supports a role of c-MET activation in tumour invasive front in the tumour progression and invasion involving FAK and AKT downstream. The c-MET serves as an attractive therapeutic target in SCLC, as shown through small interfering RNA (siRNA) and selective prototype c-MET inhibitor SU11274, inhibiting the phosphorylation of c-MET itself and its downstream molecules such as AKT, S6 kinase, and ERK1/2. Investigation of mechanisms of invasion and, ultimately, metastasis in SCLC would be very useful with these signal transduction molecules.

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“…Phosphoscreen on TA muscle Whole cell lysates were prepared from samples of unstretched and stretched TA (tibialis anterior) muscle obtained from juvenile (8-week-old) C57 and γ SG -/-mice according to published protocols (Pelech et al, 2003). Stretching of ~10% for 20 minutes was done after dripping calcium-free PBS on the muscle.…”

Section: Single Cell Relaxationmentioning

confidence: 99%

“…Stretching of ~10% for 20 minutes was done after dripping calcium-free PBS on the muscle. 300 µg of these protein lysates were separated by SDS-PAGE and were immunoblotted in a 20-lane Immunetics multiblotter using commercially available antibodies as recently described for the Kinexus KPSS5.0-phospho and KPSP-1 phosphoscreen (Pelech et al, 2003). The immunoblots were then subjected to enhanced chemiluminescence, and were identified based upon human protein sequences.…”

Section: Single Cell Relaxationmentioning

confidence: 99%

γ-Sarcoglycan deficiency increases cell contractility, apoptosis and MAPK pathway activation but does not affect adhesion

Griffin

Feng

Tewari

et al. 2005

Journal of Cell Science

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The functions of γ-sarcoglycan (γSG) in normal myotubes are largely unknown, however γSG is known to assemble into a key membrane complex with dystroglycan and its deficiency is one known cause of limb-girdle muscular dystrophy. Previous findings of apoptosis from γSG-deficient mice are extended here to cell culture where apoptosis is seen to increase more than tenfold in γSG-deficient myotubes compared with normal cells. The deficient myotubes also exhibit an increased contractile prestress that results in greater shortening and widening when the cells are either lightly detached or self-detached. However, micropipette-forced peeling of single myotubes revealed no significant difference in cell adhesion. Consistent with a more contractile phenotype, acto-myosin striations were more prominent in γSG-deficient myotubes than in normal cells. An initial phosphoscreen of more than 12 signaling proteins revealed a number of differences between normal and γSG–/– muscle, both before and after stretching. MAPK-pathway proteins displayed the largest changes in activation, although significant phosphorylation also appeared for other proteins linked to hypertension. We conclude that γSG normally moderates contractile prestress in skeletal muscle, and we propose a role for γSG in membrane-based signaling of the effects of prestress and sarcomerogenesis.

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Kinetworks<sup>TM</sup> Protein Kinase Multiblot Analysis

Cited by 35 publications

References 3 publications

B23/Nucleophosmin Serine 4 Phosphorylation Mediates Mitotic Functions of Polo-like Kinase 1

B23/Nucleophosmin Serine 4 Phosphorylation Mediates Mitotic Functions of Polo-like Kinase 1

Downstream signalling and specific inhibition of c-MET/HGF pathway in small cell lung cancer: implications for tumour invasion

γ-Sarcoglycan deficiency increases cell contractility, apoptosis and MAPK pathway activation but does not affect adhesion

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