Journal: Journal of Cell Science

Article Title: In vitro reconstitution of a minimal human centrosome scaffold capable of forming and clustering microtubule asters

doi: 10.1242/jcs.264121

Figure Lengend Snippet: Phospho-regulation of in vitro CDK5RAP2 scaffolds. (A) GFP::CDK5RAP2 assemblies generated in the presence of purified kinase dead (KD) or constitutively active (CA) human PLK-1. Scale bars: 5 μm. (B) Quantification of results as in A. Data points represent masses of individual GFP::CDK5RAP2 assemblies [mean±95% c.i.; KD PLK-1 condition ( n =604 assemblies), CA PLK-1 condition ( n =511 assemblies)]. Significant differences were assessed using a Mann–Whitney test. (C) Total number of assemblies per image from experiments as in A. Each data point represents the total number of assemblies per image (mean±95% c.i.; KD n =7, CA n =7). Significant differences were assessed using a Mann–Whitney test. (D) Identified PLK-1 phosphorylation sites (p-Sites) in GFP::CDK5RAP2 incubated with human PLK-1 plus ATP·MgCl2. Control reactions consisted of pre-dephosphorylated GFP::CDK5RAP2 plus PLK-1 but no ATP·MgCl2. (E) In vitro assemblies of pre-dephosphorylated GFP::CDK5RAP2(WT) and GFP::CDK5RAP2(S613E) without PLK-1 or ATP·MgCl2. Scale bars: 5 μm. (F) Quantification of results as in E. Each data point represents the mass of an individual GFP::CDK5RAP2 assembly [mean±95% c.i.; WT ( n =1337 assemblies), S613E ( n =2463 assemblies)]. Significant differences were assessed using a Mann–Whitney test. (G) Total number of assemblies per image from results as in E. Each data point represents the total number of assemblies per image (mean±95% c.i.; WT n =7, S613E n =7). Significant differences were assessed using a Mann–Whitney test. ** P <0.01; **** P< 0.0001; ns, not significant. a.u., arbitrary units.

Article Snippet: All samples were then immediately transferred to a glass slide and imaged at 37°C using a TokaiHit incubation chamber.

Techniques: In Vitro, Generated, Purification, MANN-WHITNEY, Phospho-proteomics, Incubation, Control

Journal: Microarrays

Article Title: Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays

doi: 10.3390/microarrays5020016

Figure Lengend Snippet: Evaluation of slide-based solid supports for antibody microarrays. The supports were compared with respect to blocking buffer, surface fouling, spot morphology and signal strength. ( A ) The slides (no antibodies printed) were first blocked and then incubated with crude, labelled serum and scanned after washing. Any observed signal intensity represents non-specific background binding. Representative scans of FAST, black MaxiSorp, Nexterion H, epoxy polymer and NHS glass slides are shown. The blocking agents tested are shown to the right; ( B ) Scanned microarray images of 14 × 8 antibody microarrays on epoxy glass, epoxy polymer, NHS glass, Nexterion H, black MaxiSorp, GAPSII, Nexterion P, Silane-Prep and NHS polymer slides. The printed antibody arrays were blocked and then incubated with crude, labelled serum before scanning.

Article Snippet: For manual processing, individual sub-arrays were created by using silicon incubation chambers (Schott) or a hydrophobic pen (Dako, Glostrup, Denmark) for slides that did not fit the incubation chambers (Silane-Prep (Sigma) and GAPSII (Corning)).

Techniques: Blocking Assay, Incubation, Binding Assay, Polymer, Microarray