polyclonal rabbit anti eg5 antibody nb500 181  (Novus Biologicals)

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5

doi: 10.1002/cm.21380

Figure Lengend Snippet: A. Two-color Western blot showing endogenous Eg5 immunoprecipitated from HEK293T or LLC-Pk1 cell lysates, with each channel displayed separately in black and white. A-419259 was added as indicated; the lower panel shows a β-tubulin loading control (green). B. The structure of Eg5 bound to S-trityl-L-cysteine (STLC) is marked with tyrosines Y125, Y211, and Y231 (orange space fill, PDB: 3KEN). A predicted SH3 binding site in the MT-binding site of the Eg5 motor domain is shown in the inset. L5 is shown in dark blue; Loop 12 within the MT binding domain is shown in red. C. Sequence alignment comparing the putative phosphorylation sites and the –PXXP– SH3 targeting domain. Putative phosphorylated tyrosines and the –PXXP– SH3 targeting motifs are shown in red. The accession numbers for each protein are listed in Fig. S1 C.

Article Snippet: For detection of endogenous Eg5 we used 1:5000 polyclonal rabbit anti-Eg5 NB500-181 antibody (Novus Biologicals, Littleton, CO).

Techniques: Western Blot, Immunoprecipitation, Control, Binding Assay, Sequencing, Phospho-proteomics

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5

doi: 10.1002/cm.21380

Figure Lengend Snippet: Kinesin-5 constructs (denoted across the top) were incubated with human c-Src (A) or human Wee1 kinase (B) and radiolabeled ATP for the times indicated. Reactions were quenched, run on an SDS-PAGE (top) that was then dried and exposed to film (bottom). Position of c-Src, Eg5, and Wee1 marked on the left side. C. Immunoprecipitation of Eg5 from HEK293T cells co-transfected with myc-tagged Eg5 motor head and c-Src constructs; A-419259 added as indicated. Western blot stained using anti-myc and anti-pTyr antibodies which here are displayed separately in black and white. The lower panel shows inputs and β-tubulin level as a loading control. D. Endogenous Eg5 was immunoprecipitated from HEK293T cells transfected with the indicated constructs; A-419259 was added as indicated. pTyr was detected using a two-color Western blot. Each channel is displayed separately in black and white. Transfection efficacy was verified by detection of c-Src in the lysates from transfected cells. β-catenin level is shown as a loading control.

Article Snippet: For detection of endogenous Eg5 we used 1:5000 polyclonal rabbit anti-Eg5 NB500-181 antibody (Novus Biologicals, Littleton, CO).

Techniques: Construct, Incubation, SDS Page, Immunoprecipitation, Transfection, Western Blot, Staining, Control

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5

doi: 10.1002/cm.21380

Figure Lengend Snippet: Effects of phosphomimetic and non-phosphorylatable mutations on Eg5 motor characteristics and STLC binding Steady-state ATPase rates and MT sliding velocities of Eg5 phosphomimetic (E) and non-phosphorylatable (F) mutants were measured and compared to those of Eg5-367-WT and Eg5-367-DL5 mutants using standard in vitro assays (Methods). Dissociation constants of the L5 inhibitor STLC to Eg5-367 phosphomimetic and non-phosphorylatable mutants (K D ) was calculated from ITC titrations as described in the Methods. Errors in K D were estimated based on the nonlinear least-squares fits to raw ITC data. Stoichiometries (N) show some variability reflecting protein concentration determination, but are generally consistent with single-site binding.

Article Snippet: For detection of endogenous Eg5 we used 1:5000 polyclonal rabbit anti-Eg5 NB500-181 antibody (Novus Biologicals, Littleton, CO).

Techniques: Binding Assay, In Vitro, Protein Concentration

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5

doi: 10.1002/cm.21380

Figure Lengend Snippet: A. Percent of mitotic phenotypes in LLC-Pk1 cells transfected with siRNA targeting endogenous Eg5 alone or co-transfected with an siRNA resistant Eg5 Emerald construct (WT, Y211E, Y211F, GSTY). Monopole (yellow), bipole (red), muitipole (blue), disorganized (green). Examples of each phenotype are shown on right. B. Time-lapse imaging of LLC-Pk1 cells expressing mCherry-α-tubulin (right panels) co-transfected with Eg5 siRNA and siRNA resistant Eg5 Emerald constructs (WT, Y211E, and Y211F left panels). C. Immunofluorescence staining for MTs in control (left) and SU6656-treated (right) parental LLC-Pk1 cells. D. Quantification of mitotic spindle phenotypes shown in C. E. Immunofluorescence staining for MTs (top) and phospho-Aurora (bottom) for control and SU6656 treated cells. (F) Immunofluorescence staining of α-tubulin in anaphase LLC-Pk1 cells: control (top), BI-2536 (middle), SU6656 (bottom). ** = p ≤ 0.01. Scale bars in A, B, C, E, F = 5 μm. Time in B (min:sec). Error Bars = St Dev.

Article Snippet: For detection of endogenous Eg5 we used 1:5000 polyclonal rabbit anti-Eg5 NB500-181 antibody (Novus Biologicals, Littleton, CO).

Techniques: Transfection, Construct, Imaging, Expressing, Immunofluorescence, Staining, Control