Typhoon 9500 laser scanner

Equivalent amounts of protein from the collected fractions were separated by 12.5 % sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The separated proteins were silver stained and the gel image acquisition was performed using ArtixScan 1100 scanner (Microtek, Hsinchu, Taiwan). The proteins from similar gels were transferred to nitrocellulose membranes and analyzed by Western Blot assay. The nonspecific binding was blocked with 5 % bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.05 % Tween 20, pH 7.6. The blots were than exposed for 2 h to the primary anti-caveolin-1 (0.25 μg/ml working concentration), anti-PTRF (1 μg/ml final concentration), or anti-beta actin (0.5 μg/ml working concentration) antibodies in TBS with 1 % BSA. Incubation for 1 h with appropriate secondary IgG-HRP antibodies was performed and the chemiluminiscence reaction was recorded. In the case of the vinculin antigen detection, only the first monoclonal antibody (conjugated with FITC) was used. For this situation, the specific excitation/emission FITC filters were used inside the Typhoon 9500 laser scanner (GE Healthcare, Uppsala, Sweden).

The concerted integration activity of Sso7d(W24A/R43E)-HTLV-1 IN(wt) was tested using a 3′-OH recessed viral DNA substrate 25 R containing the HTLV-1 U5 LTR sequence, prepared by annealing two HPLC-purified oligonucleotides 5′-Cy5-CCAGGAGAGAAATTTAGTACACA-3′ and 5′-ACTGTGTACTAAATTTCTCTCCTGG-3’ (IDT). The reaction mix initially included 0.5 μM viral DNA substrate and 1.5 μM IN in 25 mM HEPES (pH 7.0), 100 mM NaCl, 10 mM MgCl₂, 10 µM ZnCl₂, 10 mM dithiothreitol (DTT), and 10% (v/v) dimethyl sulfoxide (DMSO). After an initial preincubation at 14 °C for 15 min, the supercoiled target DNA, pBSKZeo (2.7 kb)^{46 (link)}, was added to a final concentration of 8 nM, and strand transfer was carried out at 37 °C for 45 min. The reactions were stopped by adding EDTA to a final concentration of 25 mM, and samples were deproteinized with 0.5% SDS, 1 mg/ml proteinase K for 1 hr at 37 °C. Strand-transfer products were separated on a 1.5% agarose gel and visualized by scanning for Cy5 fluorescence on Typhoon 9500 Laser Scanner (GE Healthcare Life Sciences). The gel was stained with SYBR Gold (Invitrogen) and analyzed by a Typhoon 9500 scanner to visualize the target DNA (shown on the left and right, respectively, in Supplementary Fig. 8).

The concerted integration assays were performed using 3′-OH–recessed oligonucleotide viral DNA substrates with RSV IN as previously described (3 (link), 4 (link)). Double stranded 3′-OH–recessed substrates containing RSV gain-of-function (G) U3 and WT U3 LTR sequences were 18 nucleotides in length and synthesized by Integrated DNA Technologies. The DNA substrates were recessed by two nucleotides on the catalytic strand and designated with an R. The identified length of the oligonucleotide denotes the noncatalytic strand. The sequences were as follows: GU3 18R (5′-ATTGCATAAGACAACA-3′ and 5′-AATGTTGTCTTATGCAAT-3′). The bold underlined nucleotide on the catalytic strand was different between the GU3 and WT U3 sequence. The concentrations of IN and the viral LTR substrate in a typical assay were 2 and 1 μM, respectively. The strand transfer products were separated on a 1.3% agarose gel, stained with SYBR Gold (Invitrogen), and analyzed by a Typhoon 9500 Laser Scanner (GE HealthCare Life Sciences).
The 3′-OH processing of ³²P-labeled blunt-ended viral 4.6 kb DNA at 37 °C was previously described (32 , 39 (link)). Concentrations of purified IN and DNA in the assay mixture was 20 nM and 0.5 nM, respectively.