XB-IMG-116914
Xenbase Image ID: 116914
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Figure 2. Validation of VIP Assay(A) VIP binding isotherm of EB1-EGFP to αEB1-antibody interaction fitted with a one site–binding model. A.U., arbitrary units.(B) Interaction of EB1-EGFP (prey: EGFP protein added to the extract) with APCMTBD (bait: recombinant protein added to the extract that binds to the anti-APC–coated beads) in metaphase (M, open bars) and interphase (I, filled bars). The three pairs of bars correspond to three independent experiments carried out in different extracts. A.U., arbitrary units; exp, experiment.(C) FRET binding isotherm of recombinant XMAP215-EGFP to recombinant EB1Cy3 in CSFXB buffer (left panel) and the VIP binding isotherm of recombinant XMAP215 to EB1-EGFP in CSFXB buffer (right panel). Both isotherms are fitted with one site–binding models. A.U., arbitrary units.(D) IP of XMAP215 or EB1 (left panel) and XMAP215/EB1-EGFP VIP (right panel) in interphase and metaphase extracts. Co-precipitation of EB1 or XMAP215 in IP experiments was probed by SDS-PAGE/immunoblotting with the respective antibodies (immunoblot [IB] with XMAP215 antibody and with EB1 antibody). In the VIP experiment, endogenous XMAP215 served as bait and EB1-EGFP as fluorescent prey. In the right panel, the four pairs of bars correspond to four independent experiments performed in metaphase (M, open bars) and interphase (I, filled bars) extracts. A.U., arbitrary units; exp, experiment.(E) Left panel shows IP pull down of 2 μM GST and GST-C-EB1 using apolyclonal αGST antibody. Co-precipitation of XMAP215 was probed by SDS-PAGE/immunoblotting with the XMAP215 antibody (IB: XMAP215). Immunoblot (IB) with monoclonal αGST antibody (m-GST) shows that GST and GST-C-EB1 were added to interphase and metaphase extracts accordingly. Right panel shows the VIP experiment of EB1-GST-C-EB1Cy5 and XMAP215-GST-C-EB1Cy5 in metaphase (M, open bars) and interphase (I, filled bars) extracts. Error bars represent the s.e.m. of Image published in: Niethammer P et al. (2007) Copyright: © 2007 Niethammer et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |