Akt in DC Survival and Enhancementpark D Et Al

Akt in DC survival and enhancementPark D et al.

Supplementary Methods

Preparation of plasmids and recombinant adenovirus.

For the construction of Src-, Fyn- or Lck-myristoylated Akt or EGFP, annealed oligonucleotide duplexes encoding Src, Fyn or Lck myristoylation targeting sequences (Src; 5MyrS17: 5’-ggccaccatgggtagcaacaagagcaagcccaaggatgccagccag cggcgccgcagcc-3’, 3MyrS17 : 5’-tcgaggctgcggcgccgctggctggcatccttgggcttgctcttgttg ctacccatggtggccgc-3’, Fyn; 5MyrL17: 5’-ggccaccatgggctgtgtctgcagctcaaaccctgaag atgactggatggagaacattc-3’, 3MyrL17: 5’-tcgagaatgttctccatccagtcatcttcagggtttgagctg cagacacagcccatggtggccgc-3’, Lck; 5MyrF17: 5’-ggccaccatgggctgtgtgcaatgtaaggataa agaagcaacaaaactgacggaggagc-3’, 3MyrF17: 5’-tcgagctcctccgtcagttttgttgcttctttat ccttacattgcacacagcccatggtggccgc-3’) were subcloned into expression vector, pBJ5 (Spencer ’93) at Sst II and Xho I. Fragments encoding Sal I-linkered EGFP or Sal I-linkered Akt (lacking its PH domain), which were removed from plasmids pSH1/M-∆Akt-E (Li, B. et al.Gene Therapy (2001) 9: 233-244), were subcloned in-frame into the Xho I and Sal I sites downstream of each myristoylation-targeting sequence. To generate adenovirus, Ad-MF-∆Akt, expressing doubly acylated Akt, MF-Akt fragments were isolated from pBJ5-MF-Akt by Not I/Klenow and EcoR I digestion and subcloned into Sma I/EcoR I-digested shuttle vector pDNR-CMV to create pDNR-MF-Akt. Finally, recombinant adenoviral vector, pLP-Adeno-X-MF-Akt, encoding MF-Akt, was generated by Cre-loxP-mediated recombination according to the Adeno-X-Expression System 2 (BD Biosciences) protocol. To generate adenovirus expressing MF-∆hAkt (Ad5f35-MF-human ∆Akt1), human Akt1 (lacking its PH domain) fused with myristoylation targeting sequence (MF) was subcloned into shuttle vector, pShuttle-X, sequenced, I-Ceu I/I-Sce I-digested and transferred into similarly digested Ad5f35 (A. Davis, BCM Gene Vector Lab). Generation, purification and titration of recombinant adenoviruses expressing MF-Akt (Ad-MF-Akt) and MF-h∆Akt (Ad5f35-MF-h∆Akt) were performed as described in the manufacturer’s protocol (BD Biosciences).

Preparation of siRNAs. The previously identified small interfering RNA (siRNA) for Akt-1, matching closely all isoforms (siAKTc; Katome, T., et al.J. Biol. Chem. (2003) 278: 28312), was synthesized from Integrated DNA Technologies (Coralville, IA), and negative control siRNA was purchased from Ambion (Austin, TX). Transfection of siRNAs into BMDCs was done by GeneSilencerTM siRNA Transfection (GTS, San Diego, CA) or nucleofection using the Human Dendritic Cell Nucleofector Kit (Amaxa Biosystems, Gaithersburg, MD).

NF-B SEAP (secreted alkaline phosphatase) reporter assay. Jurkat TAg cells were transfected with indicated amounts of plasmids along with NF-B SEAP reporter plasmid by electroporation. The following day, cells were plated with sub-optimal concentrations of phorbol ester PMA (0.5 nM) and further incubated for ~24 hr. Thereafter, supernatants were collected and analyzed for SEAP activity as described previously (Li, B. et al.Gene Therapy (2001) 9: 233-244).

Fluorescent confocal microscopy. 293 cells cultured on poly-L-lysine-coated coverslips were transfected with indicated plasmids in 6-well plates and further incubated for 24 hr. For drug-dependent lipid-raft localization, 100 nM of Rapalog (Rapa-B) was added for an additional 4 hr. After fixation with 4% paraformaldehyde for 30 min, cells were labeled with cholera toxin B (CTx-B-TRITC) to detect glycosphingolipid in membrane lipid-rafts (List Biological Laboratories, Campbell, CA), and analyzed by LSM 430 confocal microscopy.

-1-