Whether our approach based on the use of the whole phosphorylated activation section as antigen might be generalized to additional protein kinases including CDK6 remain to be explored. Our present antibodies allow the direct detection of endogenous phospho-CDK4 from human being and mouse cells by a variety of techniques including immunoblotting, immunoprecipitation and immunofluorescence (the second option depending on overexpression). specifically recognize the active CDK4 phosphorylated on its T172 residue. One key to this success was the immunization with a long phosphopeptide related to the complete activation section of CDK4. These monoclonal antibodies specifically identify T172-phosphorylated CDK4 in a variety of assays, including western blotting, immunoprecipitation and, like a capture antibody, a sensitive ELISA from cell lysates. The specific immunoprecipitation of T172-phosphorylated CDK4 allowed to clarify the involvement of phosphorylations of co-immunoprecipitated p21 and p27, showing a privileged connection of T172-phosphorylated CDK4 with S130-phosphorylated p21 and S10-phosphorylated p27. Abbreviations: 2D: two-dimensional; CAK: CDK-activating kinase; CDK: cyclin-dependent kinase; HAT: Hypoxanthine-Aminopterin-Thymidine; FBS: fetal bovine serum; IP: immunoprecipitation; ID: immunodetection; mAb: monoclonal antibody; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffer saline; pRb: retinoblastoma susceptibility protein; SDS: sodium dodecyl sulfate; DTT: dithiotreitol; TET: tetracyclin repressor; Avi: Avi tag; TEV: tobacco etch disease cleavage site; EGFP: enhanced green fluorescent protein; BirA: bifunctional protein biotin ligase BirA; IRES: internal ribosome access site; HIS: poly-HIS purification tag; DELFIA: dissociation-enhanced lanthanide fluorescent immunoassay; 3-MBPP1: 1-(1,1-dimethylethyl)-3[(3-methylphenyl) methyl]-1H-pyrazolo[3,4-d] pyrimidin-4-amine; BSA: bovine serum albumin; ECL: Enhanced chemiluminescence the NAV-2729 same proteic fusion comprising the T172A mutation (observe below). They were then tested by western blotting from one and two-dimension electrophoresis gels. Hybridomas of interest were subcloned by limiting dilution and the acquired subclones were tested similarly as explained above for the parental clones. Two of them, NB8-AD9 and LA2-AD4, were selected for this study and were purified on Protein A-Sepharose. Both mAbs were determined to be IgG1. Table 1. Phospho-peptide sequences EGT578 is definitely a typical 13 aa-long immunizing peptide, while EGT577 is definitely 28 aa-long that corresponds to the full activation section (T-loop) of CDK4 the same Rftn2 proteic fusion comprising the T172A mutation (a), or immunoblotting detection of 2D electrophoretic gels of CHO cells that were transfected using cyclin D3 and WT or T172A CDK4 constructs (b). In (a), the normalized fluorescent devices were the ratios between the signal acquired with the fusion exposed from the sera divided by the one acquired with the fusion recognized by a cyclin D3 antibody. In (b), the positions of T172-phosphorylated CDK4 are demonstrated by arrows. Hybridoma supernatants screening and selection of mAbs Three hybridoma fusions were acquired for M1, all phospho-specific in indirect ELISA (Number S1), and 23 hybridoma fusions for M4, with 16 showing partial or total phosphospecificity. Further screening of the hybridoma fusion tradition supernatants with the DELFIA assay showed (Number NAV-2729 S2(a)) that (i) from your three M1 hybridoma fusions, only one, GB4, was positive but not phosphospecific; (ii) from your 23 M4 hybridoma, 9 were positive and more or less phospho-specific, LA2, NB8, MD8 and RF7 showing the best reactions. It should be mentioned that some supernatants like RF5 were only positive in the DELFIA assay. In parallel, the hydridoma fusion tradition supernatants were tested on immunoblots acquired by 1D-gel separation of MCF7 cells expressing a WT (132) or T172A (133) cyclin D3/CDK4 fusion or acquired by 2D-gel separation of CHO cells transfected to express WT or T172A CDK4 and cyclin D3. GB4, JE3, LA2, LC7, MD8, NB8, RC4 and RF7 showed phospho-specificity within the cyclin D3/CDK4 fusion in 1D-gel immunoblots (Number S2(b)) and in 2D-gel immunoblots of CDK4 transduced with cyclin D3 in CHO cells (Number S2(c)). NB8 NAV-2729 and LA2 were successfully cloned providing respectively two different and 14 identical clones. GB4 also yielded two apparently identical clones. NB8-AD9, NB8-CF1, LA2-AD4 and GB4-AF9 were produced (Number 4). Open in a separate window Number 4. CDK4 T172-phosphospecificity of cloned mAbs. Lysates from MCF7 cells expressing a cyclin D3/CDK4 (WT) proteic fusion or the same proteic fusion comprising the T172A mutation (T172A) were immunoprecipitated (IP) with four different phosphoT172-CDK4 mAbs (NB8-AD9, NB8-CF1, LA2-AD4 and GB4-AF9) or cyclin D3 mAb and separated by SDS-PAGE. The arrow shows the position of the cyclin D3/CDK4 fusions immunodetected having a CDK4 antibody. Characterization of purified antibodies NB8-AD9 and LA2-AD4 Immunoblotting experiments showed that NAV-2729 NB8-AD9 and LA2-AD4 detect endogenous T172-phosphorylated CDK4 from human being 16-h FBS-stimulated T98G cells or breast tumor cell lines (Number 5(a-c)).
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