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. 2007 Oct 29;204(11):2615–2627. doi: 10.1084/jem.20070318

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Copyright © 2007, The Rockefeller University Press

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Figure 1. — The identification of a new splice variant of CYLD. (A) Schematic representation of exons 6–9 of the CYLD gene, with respective transcripts. (B) RT-PCR of cDNA generated from MEFs of the indicated genotypes using primer pair P1 and P2. WT full-length CYLD transcript corresponds to the 770-bp band, whereas the alternatively spliced CYLD transcript is 244 bp. Bands were isolated, subcloned, and verified by sequencing. The intermediate band could be determined as an unspecific band. (C) Western blot analysis of lysates from WT tissue and cells. (lane 1) Spleen; (lane 2) T cells; (lane 3) LNs; (lane 4) thymus; (lane 5) B cells. Actin was used as loading control. (D) Western blot analysis of lysates from WT and CYLD^ex7/8 B cells using anti-CYLD antibody. Actin was used as loading control. (E) Schematic representation of the different CYLD constructs used for transfection experiments in HeLa cells. Highlighted are the important domains of CYLD. FL-CYLD (full-length CYLD), C/S-mut CYLD (full-length CYLD carrying a mutation from C to S to generate a catalytically inactive form), and sCYLD alternatively spliced CYLD lacking exons 7 and 8. (F) HeLa cells were transfected with plasmids encoding either His-FL-CYLD or for His-sCYLD together with either plasmid pcDNA or plasmid encoding for TRAF2. Transfected cells were lysed and coimmunoprecipitated with anti-His antibody for CYLD, separated on an 8% SDS-PAGE, and transferred to PVDF membrane. The membranes were incubated with TRAF2 and CYLD antibodies, respectively. (G) MEFs were transfected with TRAF2-FLAG cDNA and TRAF2 was immunoprecipitated with anti-FLAG antibody. Thereafter, TRAF2 was separated on a 4–12% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with Ub-specific antibody. (lane 1) Mock-transfected WT MEFs; (lane 2) WT MEFs transfected with TRAF2-FLAG; (lane 3) CYLD^ex7/8 MEFs transfected with TRAF2-FLAG. Data presented are representative of three different experiments. (H) HeLa cells were transfected with the indicated CYLD construct encoding plasmids together with plasmids coding for Flag-Bcl-3 and His-Ubiquitin. Protein lysates of transfected cells were used for immunoprecipitations with anti-His antibody, and Western blots were incubated with antibodies against the His and the Flag tags. Flag-Bcl-3 was used as a loading control for the Bcl-3 levels in the different cell types.