Ment, were performed applying PyMOL (https://www.pymol.org, accessed on 2 April 2020). Colour schemes are formated as in (A).Cancers 2021, 13,ten of Figure two. mRNA 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 specific expression in pancreas (high), lung (median), spleen (low), brain, colon and stomach (extremely 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 considerable tissue specificity in mice (C) and only a modest down regulation in some human PDAC cell lines when compared with pancreatic tissue. All mRNA expressions levels have been normalized by the HPRT housekeeping gene. p 0.001, unpaired Student’s t-test.3.3. RBPJL Does not Interact with all the Coactivator NICD Transcription factor RBPJ is known to interact not just with DNA but additionally with the NICD and mechanistic particulars have been studied in good detail not just structurally but in addition 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 also for more 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 does not interact with NICD (Figure 3A, ideal), KyoT2 (Figure 3A, right) or RITA (Figure S4A, appropriate). As a positive control, we used PTF1a, which was previously described as strongly interacting with RBPJL. This was also the case in our co-immunoprecipitation experiments: Each RBPJ and RBPJL had been capable to interact with PTF1a (Figure S4B). Importantly, both RBPJ and RBPJL also showed an interaction together with the corepressor SHARP (Figure 3C). In summary, differently from RBPJ, RBPJL doesn’t interact with all the classical RAM-like binding partners NICD, KyoT2 or RITA but does interact together with the Notch corepressor SHARP.Cancers 2021, 13,11 ofFigure 3. RBPJL doesn’t 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, Bromonitromethane custom synthesis suitable) and KyoT2 (B, appropriate). (C) RBPJL interacts with corepressor SHARP (suitable) and with RBPJ (left). HEK293 cells were cotransfected with Flag-taggedCancers 2021, 13,12 ofRBPJ or RBPJL together with all the indicated GFP-tagged constructs: NICD (which corresponds for the human NOTCH1 intracellular domain, aa 1761-2555), KyoT2 and SHARP (aa 2776-2833 correspond towards the RBPJ interaction domain, RBPID). CoIPs have been performed 24 h immediately after transfection. Immunoprecipitation was performed applying an anti-Flag antibody and coimmunoprecipitated proteins have been detected by using an anti-GFP antibody (upper panels). Expression of proteins was verified by Western blotting (middle panels and reduced panels). Original blots see Figure S8.To additional characterize the molecular mechanism of RBPJL action, we took benefit on the combined structural and functional data 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 plus the doub.