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. 2002 Mar;22(6):1778–1791. doi: 10.1128/MCB.22.6.1778-1791.2002

FIG. 1.

FIG. 1.

L27N domain of mLin-2/CASK is a novel protein-protein interaction domain. (A) (Top) Schematic of the recognized protein interaction domains in mLin-2/CASK, showing the L27 domains between the CKII and PDZ domains. mLin-2/CASK consists of a CKII domain, a PDZ (PSD-95/Dlg/ZO-1) domain found in other MAGUKs, an Src homology domain (SH3), the Hook binding domain, which mediates cytoskeletal attachment, and a GUK domain. (Bottom) Alignment of the deduced amino acid sequences of MAGUKs including mLin-2/CASK, D. melanogaster (dm) Camguk, PALS2/Vam-1, and mLin-7, which revealed two adjacent regions of high homology, the L27N and L27C domains (8). mm, mammalian. (B) GST fusion protein affinity precipitation assay using L27 domains of mLin-2/CASK, mLin-7, and PALS2. HEK 293T cells were transiently transfected with plasmids, pRK5Myc-mLin-2/CASK, and pRK5Myc-mLin-7. Proteins collected from HEK 293 cell lysates were precipitated with various GST fusion proteins including GST, GST-mLin-2/CASK-L27N+L27C, GST-mLin-7FL, GST-PALS2-L27N+L27C, GST-mLin-2/CASK-L27N, GST-mLin-2/CASK-L27C, GST-mLin-7-L27, and GST-PALS2-L27N. Precipitated proteins were detected by an anti-Myc antibody. One-tenth of the lysates used for precipitation was run as input control (Lysate). (C) Far-Western overlay assay of different cell lysates and immunoprecipitated protein complexes with a 32P-labeled GST-mLin-2/CASK L27N protein probe. Triton X-100-extracted cell lysates collected from human glioblastoma cell line A-172 (lane 1), mouse brain (lane 2), an MDCK cell line stably expressing Myc-mLin-2/CASK (lane 3), proteins precipitated with preimmune serum (lane 4), proteins precipitated with an anti-Myc antibody (lane 5), and proteins precipitated with an anti-mLin-2/CASK antibody (lane 6) were separated by SDS-PAGE and transferred to nitrocellulose. The membrane was then probed with a 32P-labeled GST-mLin-2/CASK L27N protein probe. (D) Far-Western overlay assay of lysates immunoprecipitated with a preimmune or anti-Myc antibody from MDCK cells stably expressing full-length (FL) Myc-mLin-2/CASK and Myc-mLin-2/CASK ΔL27N. Top, presence of each of the Myc-tagged mLin-2/CASK proteins; bottom, coprecipitated unknown protein that binds to the L27N domain of mLin-2/CASK. Relative molecular masses in kilodaltons are shown to the left. IP, immunoprecipitation.