Biotin hpdp

Metabolic labeling of newly transcribed RNAs was performed as described previously [42 (link), 43 ]. PANC-1 cells were incubated with 100 μM 5, 6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) for 3 h to block Pol II transcription. Transcription recovered after DRB release and newly transcribed RNAs were labeled with 200 μM 4sU. After terminating transcription with TRIzol treatment, 200 μg total RNAs were used for biotinylation and purification of 4sU-labeled nascent RNA. After incubating with 0.2 mg/mL EZ-link biotin-HPDP (Pierce, 21341, dissolved in dimethylformamide (DMF, Sigma, D4551) at a concentration of 1 mg/mL) in biotinylation buffer (10 mM Tris PH 7.4, 1 mM EDTA) at room temperature (RT) for 1.5 h with rotation, bound biotin-HPDP were purified with chloroform and precipitated using equal volume of isopropanol and 1:10 volume of 5 M NaCl. Biotinylated 4sU-labeled RNAs were isolated using 150 μL streptavidin-coated magnetic beads (Invitrogen) for 30 min at RT. After substantially washed on beads, nascent RNAs were eluted twice with 100 μL 0.1 M dithiotheitol (DTT) and precipitated with 40 μL of 4 M LiCl, 2 μL glycogen, and 600 μL ice-cold ethanol.

The assay was carried out as described previously. Briefly, cells were homogenized in HEN buffer [250 mM HEPES-NaOH (pH 7.7), 1 mM EDTA, and 0.1 mM neocuproine] supplemented with 100 μM deferoxamine and centrifuged at 13,000 g for 30 min at 4°C. Cell lysates (240 μg) were added to the blocking buffer [HEN buffer adjusted to 2.5% SDS and 20 mM methyl methanethiosulfonate (MMTS)] at 50°C for 20 min with frequent vortexing. The MMTS was then removed by acetone and the proteins were precipitated at -20°C for 20 min. After acetone removal, the proteins were resuspended in HENS buffer (HEN buffer adjusted to 1% SDS). To the suspension was added to 4 mM biotin-HPDP in dimethyl sulfoxide without ascorbic acid. After incubation for 3 h at 25°C, biotinylated proteins were precipitated by streptavidin-agarose beads, which were then washed with HENS buffer. The biotinylated proteins were eluted by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and subjected to Western blot analysis. EDTA, neocuproine, deferoxamine, SDS, MMTS, biotin-HPDP, and streptavidin-agarose beads were purchased from Sigma-Aldrich (Sigma).