for 20 min and washed with PBS without Ca2+ and Mg2+. Cells were blocked at 25uC for 10 min with Goat Serum (3%) in PBS without Ca2+ and Mg2+. Cells were incubated with primary antibody (1:200) in Goat Serum (3%) for one hour, in the dark, at 37uC. Cells were washed 36 with PBS without Ca2+ and Mg2+ and secondary antibodies were added (1:50) in Goat Serum (3%) for one hour, in the dark, at 37u. Cells were washed 36 with PBS without Ca2+ and Mg2+ and a sufficient volume of DAPI working solution was added to completely cover the sample, incubating for 2–10 min, in the dark, at 25uC. Coverslips were washed to remove excess DAPI and mounted onto a slide with Fluoromount G (10 ml, Southern Biotech, Birmingham, AL). Cells were imaged with a Zeiss LSM 710 Confocal System (George Washington University, Center for Microscopy and Image Analysis). Western Blotting Cells were collected from culture and spun at 1,800 rpm, for 5 min, at 4uC, and pellets were washed twice with phosphate buffered saline (PBS) without Ca2+ and Mg2+ (Quality Biolog- ical). Cell pellets were resuspended in lysis buffer (50 mM Tris- HCl, pH 7.5, 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM Na3VO4, 1 mM DTT, one complete protease cocktail tablet/50 mL) and incubated on ice for 20 min, with gentle vortexing every 5 min. Cell lysates were centrifuged at 10,000 rpm for 10 min. Protein concentrations were determined using Bradford protein assay (Bio-Rad, Hercules, CA). Cell extracts were loaded (50–75 mg) on a 4–20% SDS- PAGE gel, run at 200 V, and transferred onto nitrocellulose membranes. Membranes were blocked with PBS containing 0.1% Tween-20 and milk (5%), and incubated overnight at 4uC with the appropriate primary antibody. Membranes were incubated with the appropriate secondary antibody and developed next day. Whole cell extracts from CD4+ primary T-cells were prepared using M-PER Mammalian Protein Extraction Reagent (Pierce, USA). Cell debris was cleared from the lysates by centrifugation (5 min, 14,000 g). Protein concentrations were determined using Bradford protein assay (Bio-Rad, Hercules, CA). Cell lysates were resolved by SDS-PAGE and transferred to a PVDF membrane. The blots were blocked for 1 h with Odyssey blocking buffer (Licor, Lincoln, NE) and rinsed with PBST (PBS +0.05% Tween- 20), once for 10 min and twice for 5 min. The membranes were then incubated with either a-Drosha (1:1000) or a-Actin (1:2000) monoclonal antibodies (Abcam, Cambridge, MA), for 2 hours at room temperature, rinsed with PBST (once for 10 min and twice for 5 min), and incubated for 1 hour with an a-rabbit and a-mouse secondary antibody (Licor, Lincoln, USA) conjugated to IRDy800 and IRDy680 respectively. Signals were detected by Licor Pearl Pulse Imager. Densitometry was performed using ImageJ software (Wayne Rasband, NIH, USA). Proteasome Inhibitor Treatments C81 HTLV-1 infected cells were plated at a density of 16106 cells/well in a 12-well plate. Cells were treated with three concentrations (0.1, 1.0, and 10 mM) of the proteasome inhibitors ALLN (208750), and PSI (539160) obtained from EMD4Bios- ciences (San Diego, CA). Cells were collected 48 hours post treatment, washed, lysed, and Western blotted for Drosha and b- Actin. Alternatively, 293T cells were treated with 10 mM of PSI and 24 hours later were transfected with the indicated expression vectors (pACH.Tax or pACH.M22). Cells were collected, washed, lysed, and prepared for immunoprecipitation. HTLV-1 Tax and Drosha Interactions RT-PCR Total RNA was isolated from the indicated cell types using the TRIzol protocol (Invitrogen, Carlsbad, CA). From total RNA, 2 ml of RNA was used for the cDNA synthesis reaction using iScript (Bio-Rad, Hercules, CA). The newly synthesized cDNA was used as the template for a PCR reaction for Drosha (TGCAACTGG- TAGCCACAGAG, ACACTGCTGAAGCTGGGATT) and GAPDH (GGAAGGTGAAGGTCGGAGTCAA, CCTTGACGGTGCCATGGAAT). Expression of miR326 was determined by the quantiMiR PCR kit (SBI, Mountainview, CA) using the miR326 detection (sense) primer: CCTCTGGGCCCTTCCTCCAG. Primary transcripts encoding miR326 were detected by RT-qPCR (CACCAACATCC- TAGCCCAAACG, AAGTGAGAACCGGGAAGCAAG). Immunoprecipitation Cell extracts from 293T cells alone, cells transfected with pACH.Tax (5 mg), or pACH.M22 (5 mg) were collected as described above. Whole cell extracts (,1 mg) were incubated overnight, rotating, at 4uC with a-IgG, a-Drosha, or a-Tax. The next day, 50 ml of a 30% slurry of Protein A+G beads (Calbiochem, Rockland, MA) was added to the IPs and incubated for two hrs, rotating, at 4uC. The IPs were spun briefly and beads were washed 16 with TNE150+0.1% NP-40, followed by a 16 wash with TNE50+0.1% NP-40. Proteins were eluted off of the beads with Laemmli buffer, run on a gel, and western blotted for both Drosha and Ubiquitin. Reverse Transcriptase Assay Supernatants were collected at indicated time points to test for the presence of virus. Viral supernatants (10 mL) were incubated in a 96-well plate with a RT reaction mixture containing 16 RT buffer (50 mM Tris–HCl, 1 mM DTT, 5 mM MgCl2, and 20 mM KCl), 0.1% Triton-X100, poly(A) (1 U/mL), poly d(T) (1 U/mL), and [3H]TTP. The mixture was incubated overnight at 37uC, 5 ml of the reaction mix was spotted on a DEAE Filtermat paper, washed four times with 5% Na2HPO4, three times with water, and then dried completely. RT activity was measured in a Betaplate counter (Wallac, Gaithersburg, MD). Data is shown as cpms (counts per minute). GST-Pulldown HeLa whole cell extracts (1 mg) were prepared and incubated with 10 ug of the following GST-Tax constructs bound to glutathione-sepharose beads: GST-Tax wildtype, GST-Tax 1– 244 (N-terminus), GST-Tax 244–336 (C-terminus), and GST-Tax 288–353. Extracts were brought up to 500 ml in TNE50+0.1% NP- 40 (100 mM Tris-HCl, pH 7.5; 50 mM NaCl; 1 mM EDTA; 0.1% NP-40) buffer and incubated overnight at 4uC with rotation. The following day, samples were spun and washed twice with TNE150+0.1% NP-40 (100 mM Tris, pH 8.0; 300 mM NaCl; 1 mM EDTA, 0.1% NP-40) buffer and 16 with TNE50+0.1% NP-40 to remove nonspecifically bound proteins. Samples were loaded and run on a 4–20% Tris-Glycine SDS-PAGE gel and subjected to Western blotting for the presence of Drosha. AntagomiR Experiments AntagomiRs were obtained from Qiagen as follows: miScript miRNA inhibitor Anti-hsa-let-7i (Catalog # MIN0000415), Anti- hsa-miR-199a-3p (Catalog # MIN0000232), and Anti-hsa-miR- 132 (Catalog #MIN0000426). AntagomiRs were transfected into 293T cells using Attractene as described above. Briefly, 293T cells were plated at 16105 cells/well in a 6 well plate. AntagomiRs were PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e40662