Bestatin

To observe Lys-acetylation of the c-MYC oncoprotein, 293 HEK cells were co-transfected with CβF-CMV-c-MYC (FLAG-tagged) in the presence of increasing amounts of CMV-HTLV-1 p30^II-GFP or an empty vector control. The cells were pelleted by centrifugation at 4° C, resuspended in RIPA buffer (phosphate-buffered saline, PBS, 1% v/v IGEPAL-CA630, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, SDS) containing protease inhibitors (antipain dihydrochloride, bestatin, leupeptin, aprotinin, chymostatin, and pepstatin (Roche Applied Sciences), and lysed by sonication at 70% duty cycle using a microprobe. Acetylated cellular proteins were immunoprecipitated using an anti-Acetyl-Lysine antibody (Millipore)/Protein G-agarose (Invitrogen), centrifuged and washed, resolved by SDS-PAGE, and the bound acetylated-c-MYC protein was detected by immunostaining as in Patel et al., 2004 (link). The status of c-MYC-acetylation in HTLV-1-transformed HuT-102 and MJG11 T-lymphocytes was determined by immunoprecipitating cellular acetylated proteins using an anti-Acetyl-Lysine antibody/Protein G-agrose. As a negative IP control, immunoprecipitations were carried out in parallel using an anti-GFP antibody (Sigma-Aldrich). The acetylated-c-MYC was detected by immunoblotting as described (Patel et al., 2004 (link)). Uninfected Jurkat E6.1 T-lymphocytes were included as a control.