Additional file 4: Figure S3. of Gibson Deletion: a novel application of isothermal in vitro recombination

Diagnostic cut related to Fig. 4. DNA isolated from blue clones was cut with KpnI or AflII and run on an agarose gel. The expected correct band pattern is reported in the inset on the right. The expected number of nucleotides deleted by Gibson Deletion (smaller deletions on top and larger deletions on the bottom) is reported on top of each group of lanes. Uncut, AflII or KpnI cut pUC19 are run on the left lanes of each gel as reported. Each clone’s DNA was cut with AflII or KpnI and the two digestions run on consecutive lanes. DNA resulting from an incorrect assembly is labelled with a X on the bottom of the two lanes corresponding to the incorrect clone. (PDF 15481 kb

Additional file 3: Figure S2. of Gibson Deletion: a novel application of isothermal in vitro recombination

Diagnostic cut related to Fig. 3. DNA isolated from clones that grew on selection plates was cut with the indicated enzymes. Group 1 cloning is depicted in Fig. 3a top panels and Group 2 cloning is depicted in Fig. 3a bottom panels. Images of simulated agarose gels are presented on the left of each cloning group. Red arrowheads indicate the clones with DNA that underwent successful/correct Gibson Deletion reaction. (PDF 3775 kb

Additional file 1: of Gibson Deletion: a novel application of isothermal in vitro recombination

The sequences of the oligonucleotides used in this study are reported. (XLSX 58 kb

Additional file 5: Figure S4. of Gibson Deletion: a novel application of isothermal in vitro recombination

Sequences of the incorrect Gibson Deletion of 200 nt related to Fig. 5. Sequences of the DNA flanking the RFP cassette (red box) cloned in pUC19 using Gibson Deletion. The RFP cassette was PCR amplified using primers with homology arms aimed to delete 200 nts (100 nucleotides from each side of a KpnI cut) from the pUC19 plasmids (Fig. 5). All the clones picked and sequences had incorrect assembly and are presented here together with the predicted sequence on top (correct). Underlined sequences are part of the RFP cassette; red sequences are sequences that are deleted after assembly using Gibson Deletion; sequences in bold are insertions obtained after assembly. (PDF 1358 kb

Additional file 1: Figure S1. of Fluorescence ImmunoPrecipitation (FLIP): a Novel Assay for High-Throughput IP

Mass spectrometry of tryptic peptides of YFP fusion proteins recovered from upper and lower bands of HES-1 expressed the HuEV-A vector. Figure S2. Mass spectrometry of tryptic peptides of YFP fusion proteins recovered from upper and lower bands of URI as in Figure S1. Figure S3. Standard curve of YFP amounts versus fluorescence reading (excitation 475 nm, emission 527 nm). Figure S4. a) HeLa cells were plated and transfected with Fugene-HD (Promega) in different size wells according with the parameters reported in the table. b) After lysis of the cells in each of the wells, the YFP fluorescence of 10 ml of solution used for IP was 2 measured. Figure S5. Tet-ON HeLa cells were transfected with the HuEV-A construct encoding the target protein with an N-terminal FLAG, YFP (Venus) and V5 tag under a tet-inducible promoter. (PDF 5435 kb

Additional file 2: Table S1. of Long interspersed nuclear element-1 expression and retrotransposition in prostate cancer cells

Molecular characteristics and origins of prostate cancer cell lines. (DOCX 43 kb

Additional file 1: Figure S1. of Long interspersed nuclear element-1 expression and retrotransposition in prostate cancer cells

Androgen receptor expression and full length of blots shown in Fig. 1a (.pdf) A) Western blot analysis of the androgen receptor (AR) in prostate cancer cell lines. Antibody against the AR recognized both full length (FL) and the V7 spliced variant. Tubulin was used as a loading control. B) High and low molecular weight regions surrounding ORF2 and ORF1. Overexposed images are from the same blot as shown in Fig. 1a. (PDF 2780 kb

URI shuttling between the cytoplasm and the nucleus is dependent on CRM1/XPO1-exportin.

<p>LNCaP cells stably overexpressing an URI-EGFP fusion protein were used to visualize URI localization (a,b and c). Cells were treated with α-amanitin (d,e and f), α-amanitin + leptomycin B (g, h and i) or with actinomycin D (j–l). Cells were counterstained with DAPI (visualized as blue fluorescence). Single colors and merged pictures are presented.</p

Additional file 3: Figure S2. of Long interspersed nuclear element-1 expression and retrotransposition in prostate cancer cells

High and low molecular weight regions surrounding ORF1 and ORF2 western blots. (.pdf) Entire length of western blots presented in Fig. 1b, showing ORF2 and ORF1 expression in cellular fractions. (PDF 329 kb

URI is part of the R2TP/prefoldin-like complex.

<p>List of nuclear URI interactors obtained from immunoprecipitation of FLAG-URI from control cells (CTRL IP) and URI overexpressing cells (URI IP). The analysis was repeated twice and the number of peptides retrieved in the two analyses are reported together with the international protein index (Accession) and the name of each protein.</p