Figure 2.
Extent of nucleosomes loading at the HO-puro locus in unselected and reselected cells, and the role of Artemis. A) Graphical sequence alignment of representative clones along the T7ST-Puro1 sequence - obtained with "Align 2" of NCBI BLAST. B) Histone H3 occupancy in unselected and reselected cell by ChiP. Chromatin was formaldheide cross-linked from 106 cells and ChIP for H3 was carried out as described in [7]. Input dilutions are from DNA isolated from 5000 cells and 3-fold dilutions thereof. PIS is the CHIP protocol carried out with pre-immune serum. ¼ input DNA is product generated by cross-link reversal from ¼ the material used for ChIP. Amp/Puro is the band generated with primers Amp1 and Puro1. C) Western blot for Artemis was carried out from reselected cells, before and after 24h treatment with siRNA against Artemis (purchased from Dharmacon). The blot was sequentially probed for Hsp90 and ATM. All antisera were from Abcam. D) Kinetics of cleavage and repair (HO time course of infection) with reselected cells treated or not with Artemis siRNA.