protein kinase identification  (Biosynth Carbosynth)

Journal: International Journal of Biological Sciences

Article Title: Identification of Novel Focal Adhesion Kinase Substrates: Role for FAK in NFκB Signaling

doi: 10.7150/ijbs.10273

Figure Lengend Snippet: Microarray screen for FAK substrates. a. Left: Autoradiograph image of control and FAK protein microarray slides. Right: Control microarray containing 1, 5, 10 and 25 ng/dot of GST or GST-Paxillin. b. Spot-pair signals from control and FAK slides block #40 (upper panels), with identified proteins, microarray positions and relative signal intensities (average pixels/pair) below. A-F, proteins with increased phosphorylation by FAK relative to control; G, positive control protein, PKCeta; H-J , proteins with equal or decreased phosphorylation levels in the FAK slide compared to control.

Article Snippet: The following reagents/kits were used: ProtoArray Human Protein Microarray v5.0 Kinase Substrate Identification Complete Kit (Invitrogen), full-length active Src (SignalChem), GST-FAK (Invitrogen), baculovirus-encoded Pyk2 (gift of Vita Golubovskaya, Roswell Park Cancer Institute [RPCI]), purified vitronectin (Advanced BioMatrix), purified GST-PTPN5[a.a.17-565] (ProteinTech), FAK inhibitor PF-573,228 (Sigma), TNFα (Abcam), Recombinant Light (Enzo Life Sciences).

Techniques: Microarray, Autoradiography, Blocking Assay, Positive Control

Journal: Nature Communications

Article Title: Identification of a novel actin-dependent signal transducing module allows for the targeted degradation of GLI1

doi: 10.1038/ncomms9023

Figure Lengend Snippet: ( a ) Autoradiograph of protein microarray showing duplicate spots of recombinant ABLIM1 without (left) or with (right) recombinant DYRK1A, both with radioactive ATP. ( b ) Co-immunoprecipitation of exogenous DYRK1A and ABLIM1 proteins in Hek293T cells. Shown is a representative blot of three independent experiments. ( c ) Cellular actin fractionation assay in NIH3T3 stably expressing empty vector control (mock) or ABLIM1. Jaspla, Jasplakinolide 10 nM for 30 min. Shown is a representative β-actin western blot of two independent experiments (the mean band intensity is given below). ( d ) Cellular actin fractionation assay in NIH3T3 stably expressing empty vector control (mock) or ABLIM2. Jaspla, Jasplakinolide 10 nM for 30 min. Shown is a representative β-actin western blot of two independent experiments (the mean band intensity is given below). ( e ) Deconvolution images of F-actin fibres (phalloidin) in NIH3T3 cells transiently expressing the indicated constructs. Transfected cells were identified through co-transfection of nuclear H2B–GFP. The F-actin promoting Profilin (PFN1) was included as a positive control. Scale bar, 10 μm. ( f ) Quantification of the microscopy experiment depicted in e (mean of two independent experiments with at least 50 cells counted in each experiment). ARR, actin-rich region (lamellipodia, stress fibres, membrane ruffles or pronounced phalloidin positivity). ( g ) Subcellular localization of MKL1-GFP (green) in transiently transfected Hek293A cells. Representative pictures are shown. Nuclei appear in blue (4,6-diamidino-2-phenylindole). Scale bar, 10 μm. ( h ) Quantification of the experiment depicted in f . C, cytoplasmic; N, nuclear; N+C, nuclear and cytoplasmic localization of MKL1-GFP (mean of n =3 independent experiments. At least 100 cells were counted). ( i ) SRE (serum response element) luciferase reporter assay in Hek293T cells transiently transfected with the indicated constructs (mean of n =6±s.d.). ( j ) Phalloidin staining of siRNA-transfected NIH3T3 cells. Shown are representative images of two independent experiments. siABlim1/2: double knockdown of Ablim1 plus Ablim2 . Scale bar, 10 μm. ( k ) Phalloidin staining of siRNA/plasmid double-transfected NIH3T3 cells. The inset in the middle panel should help portrait the different phenotypes observed upon Ablim1 knockdown. siABlim1/2: double knockdown of Ablim1 plus Ablim2. Scale bar, 10 μm. * P <0.05; ** P <0.005; *** P <0.0005 (Student's t -test).

Article Snippet: For the identification of DYRK1A protein substrates, we purchased the ProtoArray Human Protein Microarray v5.0 kinase substrate identification (KSI) kit from Invitrogen (#PAH0525065, containing ∼9,480 recombinant GST-tagged and baculovirus-expressed proteins in duplicate spots).

Techniques: Autoradiography, Microarray, Recombinant, Immunoprecipitation, Fractionation, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Construct, Transfection, Cotransfection, Positive Control, Microscopy, Luciferase, Reporter Assay, Staining