M. the levels of HEXIM1 and HIV proteins rose. Importantly, the knockdown of ELL2, a subunit of the SEC, blocked the ability of JQ1 to increase HIV transcription. Finally, the effects of JQ1 and HMBA or SAHA on the P-TEFb equilibrium were cooperative. We conclude that HMBA, SAHA, and JQ1 affect transcription Tenalisib (RP6530) elongation by a similar and convergent mechanism. for 10 min at 4 C, and supernatants were incubated with protein A-Sepharose beads for 1 h at 4 C. Beads were washed five times with 800 l of lysis buffer, and immunoprecipitated complexes were boiled in SDS sample buffer and analyzed by Western blotting. RNA Immunoprecipitations JK cells (2 106) were untreated or treated with 5 m JQ1 for 0.5 or 2 h. Cells were lysed in buffer A containing low salt (10 mm KCl) on ice for 10 min. Cell lysates were centrifuged at 5000 for 5 Tenalisib (RP6530) min at 4 C, and supernatants were collected. Supernatants were then precleared with protein A-Sepharose beads and divided into three aliquots. Each aliquot was incubated with 1 g of normal rabbit IgG, anti-HEXIM1, or anti-CDK9 antibody overnight at 4 C and then with 20 l of protein A-Sepharose beads precoated with BSA and Tenalisib (RP6530) yeast tRNA for an additional 2 h at 4 C. Beads were washed five times with medium-salt buffer A (100 mm KCl). RNA was then extracted by TRIzol (Invitrogen) and analyzed by RT-quantitative PCR (RT-qPCR). Data were normalized to input amounts of Tenalisib (RP6530) 7SK snRNA and calculated as percent values relative to the amount obtained with untreated cells (set Tenalisib (RP6530) to 100%). Differential Salt Extraction Differential salt extraction was carried out to determine fractions of free P-TEFb or 7SK snRNP according to Biglione (22) with some modifications. Jurkat cells (5 105) were collected and washed twice with cold PBS. Cells were lysed in 80 l of low-salt buffer (10 mm KCl, 10 mm MgCl2, 10 mm HEPES-KOH (pH 7.5), 1 mm EDTA, 1 mm DTT, 0.5% Nonidet P-40, and proteinase inhibitor mixture) and incubated on ice for 10 min. Lysates were then centrifuged at 5000 for 5 min, and supernatants were collected and designated as 7SK snRNP fractions. Pellets were washed once with 200 l of low-salt buffer and resuspended in 80 l of high-salt buffer (450 mm NaCl, 1.5 mm MgCl2, 20 mm HEPES (pH 7.5), 0.5 mm EDTA, 1 mm DTT, 0.5% Nonidet P-40, and proteinase inhibitor mixture). Suspensions were mixed by vortexing briefly and incubated on ice for 10 min. Lysates were then centrifuged at 10,000 for 5 min, and supernatants were collected and designated as free P-TEFb fractions. Chromatin Immunoprecipitation ChIPs were carried out according to Nelson (23) with some modifications. Briefly, Rabbit Polyclonal to MSK1 JK cells (2 107) were treated with JQ1 (5 m) or DMSO for 1 h. Cells were fixed with 1% formaldehyde in PBS for 15 min at room temperature. By adding 125 mm glycine for 5 min at room temperature, cross-linking was stopped. Sonication of chromatin was carried out using a Sonic Dismembrator 100 (Fisher) for 20 cycles of 15 s at setting 4, followed by 30 s on ice. Sheared chromatin was precleared by 50 l of protein G-Sepharose beads for 1 h at 4 C. 2 g of specific antibodies were added to the precleared lysate corresponding to 2 106 cells and incubated at 4 C overnight. Lysates were then centrifuged at 10,000 .