Ned to hg19 by Blat [61]. Alignments with e values less than
Ned to hg19 by Blat [61]. Alignments with e values less than 0.05 and that matched starting from the first nucleotide after the LTR were selected. Matches to multiple genomic sequences were removed on the basis of bit scores (differences <0.0001) to identify unique alignments. The start position of the alignment on the positive strand was chosen as the insertion site whereas 4 nucleotides were subtracted from the start position of alignments on the negative strand. To map insertions relative to TSSs, the distances between insertions and the nearest TSS were calculated and these were summed in 1.25 kb bins. If insertions were upstream of the nearest TSS the distances were plotted with negative numbers. To map insertions relative to CpG islands the inserts were given negative distances if the coordinates of an insertion were less than the coordinates of the nearest CpG. Positive distances were used if the insertions had larger coordinates than that of the nearest CpG. Bedtools was used to calculate the gene densities for each insertion [62].Immunofluorescence microscopyParental HOS cells or cells stably expressing HA-epitope tagged MxB proteins were cultured on Nunc Lab-Tek II chamber slides (Thermo Scientific). Cells were fixed with 4 paraformaldehyde for 10 min, washed with PBS, and permeabilized with ice-cold MeOH for 10 Avasimibe chemical information PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28154141 min. The cells were then blocked with blocking buffer (PBS containing 10 FBS) for 30 min, and stained with 1:300 dilution of anti-HA antibody 16b12 (Covance). After a 30 min wash with blocking buffer, the cells were incubated for 1 h with a 1:1,000 dilution of an Alexa Fluor 555 conjugated goat anti-mouse IgG antibody (Invitrogen) as well as Hoescht 33342 (Invitrogen) at 1 g/ml. After an additional 30 min wash with PBS, the samples were covered with mounting medium [150 mM NaCl, 25 mM Tris Cl pH 8.0, 0.5 N-propyl gallate, and 90 glycerol]. The processed samples were analyzed on a Nikon Eclipse spinning disk confocal microscope at the Dana-Farber Cancer Institute Confocal and Light Microscopy core.Western blottingRepresentative transcripts from sets of transcripts with identical coordinates yielded 26,251 unique RefSeq PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28242652 human genes from the UCSC Genome Bioinformatics browser [60]; 18,273 genes that yielded overlapping coordinatesCells pelleted at 300 ?g were resuspended in PBS supplemented with 0.2 NP-40 and 10 U/ml Turbo DNAse (Ambion) and incubated for 30 min on ice. Samples were mixed with protein sample loading buffer to the final concentrations of 62.5 mM Tris Cl, pH 6.8, 2 sodium dodecyl sulfate, 10 glycerol, 5 2-mercaptoethanol, and 0.001 bromophenol blue. The samples were heated forMatreyek et al. Retrovirology 2014, 11:90 http://www.retrovirology.com/content/11/1/Page 18 of5 min at 100 , separated on Tris-glycine polyacrylamide gels, and transferred to polyvinylidene fluoride membranes. MxB-HA was detected with a 1:2000 dilution of HRPconjugated 3F10 antibody (Roche), or a 1:1000 dilution of anti-MxB antibody N-17 sc-47197 (Santa Cruz Biotechnology). Beta-actin was detected with a 1:10,000 dilution of HRP-conjugated antibody clone AC-15 (Sigma). Western blots were developed using ECL prime reagent (GE Healthcare Life Sciences) and imaged with a ChemiDoc MP imager (Bio-Rad) equipped with Image Lab 4.1 software. The amount of MxB-HA or beta-actin signal in each sample was quantitated relative to the level of each signal compared to a matched WT MxB-expressing sample. The MxB expression ratio was calculate.
