Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

.... Details of the procedures are described in [51] . Immunoprecipitation Assay of p53-DNA Binding at Magnetic Beads (MBIP Assay) The Ab-p53-DNA complexes were prepared by mixing the DO1 or anti-GST antibody (400 ng) with the purified protein (50 ng) or whole cell lysate (1530 g) in binding buffer (50 mM KCl, 5 mM Tris pH 7.6 and 0.01% Triton X-100), followed by 20 min incubation on ice. Then, 300 ng of scDNA (pMSP) and/or the same amount of the linDNAs were mixed with the given immune complexes and

... by magnetic beads-based immunoprecipitation assay (MBIP) in vitro and ex vivo . A) Scheme of the procedure (from left to right): the cell lysate or purified proteins were mixed with antibody (Ab) and then with a DNA substrate and incubated to allow formation of the Ab-p53-DNA complex (a). Then, the complex is captured at DBG (magnetic beads-coated with protein G) (b). The bead suspension is washed (c), followed by dissociation of DNA from the complex with p53 (d) and DNA eluted from beads is detected by agarose electrophoresis (e, Et-Br staining). B) Control of specificity of MBIP assay for sc and lin DNAs. Binding of 100 ng of purified GST-p53CD protein to scBSK (lane 6), linBSK (lane 7), linPGM1 (lane 9) and mixtures of scBSK/linBSK (lane 8) and scBSK/linPGM1 (lane 10) by MBIP assay with anti-GST Ab are shown on the left. Lanes 15 (scBSK, linBSK, linPGM1 and sc+lin) were a control for input DNA (50 ng, 1/6 input DNA). After anti-GST immunoprecipitation, bound DNAs (lanes 615) were analyzed on 1% TAE gel (scDNA migrates faster than lin or oc forms). Binding of GST-Sp1 (construct without DNA binding domain) to different types of DNA (lanes 1115) by MBIP assay with the same Ab was not observed (right panel). C) Binding of seven purified hot spots and wtp53 to scBSK, linBSK and a scBSK/linBSK mixture by MBIP assay with DO1. Lanes 13 and 1618 are a control for input DNAs (50 ng). D) Control of p53 and actin levels in cell lysates from H1299 and H1299-R273H (induced by tetracyclin for 24 h) by WB with DO1 and anti-Actin antibody. E) Binding of R273H from H1299-R273H lysate to scMSP (lane 4), linMSP (lane 5) and a scMSP/linMSP mixture (lane 6) by MBIP assays. Lanes 13 are a control for input DNA (50 ng). Lysate from H1299 cells was used for control reaction for DO1 immunoprecipitation of sc, lin and scMSP/linMSP mixture (lanes 79). MBIP assay was performed also with recombinant purified protein R273H (100 ng) with scMSP (lane 10), linMSP (lane 11) or scMSP/linMSP mixture (lane

... Immunoprecipitation Techniques in vitro and ex vivo To analyze SCS-binding of mutp53 directly from cancer cells we used the MBIP assay (an immunoprecipitation technique, see Fig. 5A ), published recently for the determination of p53-SCS-binding by purified wtp53 [42] , [55] . Selectivity of the MBIP assay was tested with GST-p53CD (aa 92312) and GST-Sp1 (a construct lacking the DNA binding domain was used as a negative control; Fig. 5B , lanes 1115). In agreement with our EMSA experiments (C30-wtp53, Fig. 3B , lanes 47), p53 containing only core domain slightly preferred the scBSK form in competition with linBSK (GST-p53CD, Fig. 5B , lane 8). Preference for linPGM1 was observed in competition assays with scBSK by both EMSA (C30-wtp53, Fig. 3C , lanes 24) and MBIP assay (GST-p53CD, Fig. 5B , lane 10), both C30-wtp53 and GST-p53CD contains aa 92312 of core domain. MBIP assay with purified proteins confirmed our EMSA data ( Fig. 3A ) that supercoil-selective binding is a common property of all seven hot

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Gene: MBIP - MAP3K12 binding inhibitory protein 1 - (NCBI)

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