Pdsred1 n1 vector

Plasmids encoding Myc-CARMA1, Myc-CARMA1 truncated forms, EGFP-CARMA1, Myc-Bcl10, PKCθ WT, PKCθ AE [32] (link), IKKβ, and GFP-NF-κB RelA were kind gifts from Dr. Xin Lin. Expression vectors for FLAG-CARMA1 and HA-Bcl10 were generated by subcloning of coding sequence into pcDNA3 vector (Invitrogen). GST-CARMA1 CD-CC was generated by subcloning of coding sequence into pGEX-4T-1 (GE Healthcare, Buckinghamshire, UK). Human CKIP-1 cDNA was generated by PCR amplification from Jurkat cDNA and cloned into pcDNA3/hygro and pcDNA3-FLAG vector (Invitrogen). Expression vector for DsRed-CKIP-1 was generated by subcloning of coding sequence into pDsRed1-N1 vector (Clontech, Mountain View, USA). ΔLZ-CKIP-1 and ΔPH-CKIP-1 truncated form were generated by PCR amplification from WT CKIP-1 expression vector and subcloned into pcDNA3/hygro vector.

HUVEC-C3 was generated by transfecting the plasmid DNA of a FRET-based sensor C3 into HUVEC cells and isolated from a single clone19 (link). HepG2-DsRed was generated by transfecting the plasmid DNA of pDsRed1-N1 vector (Clontech, Heidelberg, Germany) into HepG2 cells and isolated from a single clone stably expressing the DsRed1 protein. All mono- and co-cultures were maintained in Dulbecco’s modified Eagle medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, USA) and 1% penicillin/streptomycin at 37 °C and 5% CO_2.