The peptide specific antibody against PLT LRSLFGND was generated by Scrum Inc. The myristoylated PKC �� peptide inhibitor myr PKC�� and myr PKC and B were purchased from Merck. Akt inhibi tor was obtained from Calbiochem, and the PI3K inhibitor Wortmannin was obtained from Merck. The Cdk inhibitor roscovitine was purchased from Promega. All inhibitors were dissolved in DMSO and selleck chemicals stocks were aliquoted and stored at ?60 C until use. The final concentration of each inhibitor used is indicated in the figure legends. Cells and viruses Monocytes were isolated from buffy coat from healthy blood donors by positive selection on Monocyte Enrich ment Cocktail and Lymphoprep density gradient centrifugation with SepMate 50. MDMs were generated by culturing monocytes with 100 ng ml granulocyte macrophage colony stimu lation factor for 5 days.

293T and HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum. HIV 189. 6 and HIV 1NLAD 8 strains were produced in 293T cells. Vesicular stomatitis virus G glycoprotein pseudotyped viruses were produced in 293T cells cotrans fected with reporter virus plasmid and VSV G using the calcium phosphate method. The culture supernatants were collected and subjected to quantification of HIV 1 particle yields by p24CA antigen capture enzyme linked immuno sorbent assay. Mono cyte isolation and treatment were approved by the Ethics Committee at the Yokohama City University School of Medicine. In vitro protein production A total of 287 cDNAs encoding human protein kinases were constructed as described previously.

The protein production method has also been described previously. Briefly, DNA templates containing a biotin liga ting sequence were amplified by split PCR using cDNAs and corresponding primers, and then used with the Gen Decoder protein production system. For HIV 1 Gag protein synthesis, Gag genes derived from the pNL4 3 proviral plasmid were generated by split PCR, and used as template with a Wheat Germ E pression kit in accordance with the manufacturers instructions. Alphascreen based protein protein interaction assays AlphaScreen assays were performed as described pre viously. All recombinant proteins used here was syn thesized using a wheat germ based cell free system as described above.

For each protein kinase, 1 ul of crude re combinant biotinylated construct from the human kinase library was incubated with 1 ul of crude GST Gag or GST DHFR in 10 ul of kinase assay buffer at 37 C for 1 h in one well of a 384 well Optiplate detection kit instruction manual, 15 ul of detection mi ture containing 100 mM Tris HCl pH 8. 0, 0. 01% Tween 20, 1 mg ml BSA, 5 ug ml Anti FLAG antibody, 5 ng streptavidin coated donor beads and 5 ng anti IgG acceptor beads were added to each well followed by incubation at Drug_discovery 26 C for 1 h.