Ment, were performed applying PyMOL (, accessed on two April 2020). Colour schemes are formated as in (A).Cancers 2021, 13,10 ofFigure 2. mRNA Lupeol Epigenetic Reader Domain expression of RBPJL (A,B) and RBPJ (C,D) in murine and human tissue samples and PDAC cell lines. (A) Relative mRNA expression of Rbpjl in tissues from C57BL/6J mice analyzed by qRT-PCR. Rbpjl shows distinct expression in pancreas (high), lung (median), spleen (low), brain, colon and stomach (very low). In the other tissues, Rbpjl mRNA is barely detectable. (B) Relative mRNA expression of RBPJL in human pancreas, PDAC and PDAC cell lines. Expression of RBPJL is downregulated in PDAC and lost in PDAC cell lines. (C,D) mRNA expression of Rbpj shows no significant tissue specificity in mice (C) and only a modest down regulation in some human PDAC cell lines in comparison to pancreatic tissue. All mRNA expressions levels were normalized by the HPRT housekeeping gene. p 0.001, unpaired Student’s t-test.three.3. RBPJL Does not Interact with the Coactivator NICD Transcription aspect RBPJ is known to interact not only with DNA but also with all the NICD and mechanistic details have been studied in terrific detail not merely structurally but also biochemically and functionally [36,37] and reviewed in [38]. The NICD contacts the BTD and CTD domains of RBPJ, and this binding surface is conserved not merely for NICD but in addition for additional cofactors KyoT2/FHL1 [39] and RITA [28]. Whereas RBPJ strongly interacts in co-immunoprecipitation experiments with NICD (Figure 3A, left), KyoT2/FHL1 (Figure 3B, left) and RITA (Figure S4A, left), RBPJL will not interact with NICD (Figure 3A, right), KyoT2 (Figure 3A, ideal) or RITA (Figure S4A, ideal). As a good control, we used PTF1a, which was previously described as strongly interacting with RBPJL. This was also the case in our co-immunoprecipitation experiments: Both RBPJ and RBPJL had been able to interact with PTF1a (Figure S4B). Importantly, each RBPJ and RBPJL also showed an interaction with the 1-Methylpyrrolidine-d3 Purity corepressor SHARP (Figure 3C). In summary, differently from RBPJ, RBPJL doesn’t interact together with the classical RAM-like binding partners NICD, KyoT2 or RITA but does interact with all the Notch corepressor SHARP.Cancers 2021, 13,11 ofFigure three. RBPJL will not interact with RBPJ “RAM”-type binding proteins (NICD, KyoT2) but does interact with corepressor SHARP. In contrast to RBPJ (left panels), coimmunoprecipitations (CoIPs) show no binding of RBPJL to NICD (A, correct) and KyoT2 (B, suitable). (C) RBPJL interacts with corepressor SHARP (suitable) and with RBPJ (left). HEK293 cells have been cotransfected with Flag-taggedCancers 2021, 13,12 ofRBPJ or RBPJL together together with the indicated GFP-tagged constructs: NICD (which corresponds towards the human NOTCH1 intracellular domain, aa 1761-2555), KyoT2 and SHARP (aa 2776-2833 correspond for the RBPJ interaction domain, RBPID). CoIPs were performed 24 h soon after transfection. Immunoprecipitation was performed applying an anti-Flag antibody and coimmunoprecipitated proteins have been detected by utilizing an anti-GFP antibody (upper panels). Expression of proteins was verified by Western blotting (middle panels and reduce panels). Original blots see Figure S8.To further characterize the molecular mechanism of RBPJL action, we took advantage in the combined structural and functional information of its paralog RBPJ [19] and also the sequence comparison of RBPJL with RBPJ (Figure 1 and Figure S1). Subsequently, we generated RBPJL mutants at the positions R220H, F262A and L393A and also the doub.