Construction of a conditional Myd88^p.L252P allele. (A) Targeting of the Myd88 locus in C57BL/6N Tac ES cells. The endogenous Myd88 locus was targeted with the linearized vector described in the supplemental Data. The targeted allele before (middle panel) and after Flp-mediated recombination of FRT and F3 sites (bottom panel) is schematically depicted. The Southern blots of BauI, Eco91I, and KpnI digested genomic DNA probed with a 5′, a 3′, and a Neo probe, respectively, are shown below the schematic illustration of the targeting strategy. Positions of restriction sites and probes are shown in the schematic drawing above. (B) Myd88^p.L252P mRNA is expressed upon Cre-mediated recombination in MEFs. Myd88^wt/wt and Myd88^{c-p.L252P/c-p.L252P} MEFs were isolated. RNA was isolated from both cell lines before LentiCre application (Sanger sequencing chromatograms, top and middle panels) and the Myd88 mRNA sequence was determined after reverse transcription. The wild-type sequence was recovered from both cell lines. After LentiCre application and puromycin selection, only the p.L252P sequence could be recovered from Myd88^{c-p.L252P/c-p.L252P} MEFs (Sanger sequencing chromatogram, bottom panel). (C) The Myd88^p.L252P isoform is expressed in Myd88^{c-p.L252P/c-p.L252P} MEFs after LentiCre-mediated recombination. Myd88^wt/wt and Myd88^{c-p.L252P/c-p.L252P} MEFs were LentiCre exposed and puromycin selected, as in (B). Whole-cell lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membranes before Myd88 and β-actin, which served as loading controls, and were visualized by immunoblotting. Both Myd88^wt and Myd88^p.L252P proteins were expressed at equal levels. (D) Conditional LentiCre-mediated Myd88^p.L252P expression leads to p65 Ser-536 phosphorylation. Myd88^wt/wt and Myd88^{c-p.L252P/c-p.L252P} MEFs were transduced with LentiCre and puromycin selected, as in (B). Upon selection, cells were lysed, proteins were separated on SDS-PAGE, and pSer-536 p65 was visualized by immunoblot. SAH, short arm of homology; LAH, long arm of homology; pA, polyadenylation signal sequence.