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For the selection procedure on plates, we rather used 500 ng/ml AHT because the growth inhibition with 1000 ng/ml AHT was too strong resulting in very low numbers of clones (not shown).
Moreover, Herceptin caused nearly a complete dephosphorylation of Akt in SK.tDp/Dp32 clones, similar to that observed in the parent SK-Br-3 cells (Fig. 3C) and SK.Neo clones (not shown).
The kinetics of development and the number of bone metastases per mouse 28 days after intra-cardiac injection of Nog-KD 17 cells did not differ from that induced by PC-3/Fluc and mock clones (not shown).
Similar results were also observed in both H460 sh-BAXBAK 1C and sh-BAXBAK4F clones (not shown).
Higher doses of gemcitabine (1 μg ml 1) led to a G1 phase synchronisation for all clones (not shown).
We also tested the half-life of 5 selected mRNAs (FAM172A, EPN, ACD, TMEM25 and ACTB) and found no differences between the three clones (not shown).
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Similarly, the galactosylated non-natural amino acid hydroxynorvalin (Gal-Hnv) peptide, which lacks aminomethylene group of hydroxylysine, failed to stimulate A8E2 hybridoma and A9.2 clone (not shown).
Human Flower isoforms, which are very similar to the mouse isoforms, were also cloned (not shown; European Patent Application number EP12156256).
However, SUM-52PE cells also have an overexpressed band at 109kDa, which was also detected in 293 cells transfected with clone C1- 38 (Fig. 5a; lane 4) and in HME cells transduced with this clone (not shown).
All cytochrome b fragments were routinely cloned (not shown), although very few heteroplasmies were detected in the direct sequencing, attributable to both the exceptional preservation of the sample and the short lengths of the fragments; consequently, the error rate in the clones (number of nucleotide differences/1,000 base pairs) was very low (< 2 per thousand base pairs).
Southern blots on 450 of these clones identified 6 recombinant clones (data not shown).
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