Rabbit anti phospho sapk jnk thr183 tyr185

To measure changes in the fraction of shRNA-containing mCherry-positive cells, cells were re-plated every 3 or 4 days. At those time points, approximately 25% of the culture was used to measure the percentage of mCherry-positive cells by flow cytometry, whereas the remaining cells were re-plated for further time points. If indicated, cells were treated with doxycycline (1 μg per mL) or ethanol as a solvent control. At least 10,000 (BT-549) or 30,000 (KBM-7) events were analyzed per sample on an LSR-II (Becton Dickinson). Cells, pre-treated with doxycycline (1 μg per mL) or HU, were harvested at different time points, washed and fixed in ice-cold 70% ethanol. Cells were permeabilized and blocked with PBS–1% BSA–0.05% Tween-20 or with PBS–2% BSA–0.1% Triton for 1 h and stained with rabbit anti-cleaved PARP (1:100, Cell Signaling, #5625), rabbit anti-phospho-SAPK/JNK (Thr183/Tyr185) (1:100, Cell Signaling, #9251), rabbit anti-TNFR1 (1:100, Abcam, #19140) or rabbit anti-γH2AX (1:100, Cell Signaling, #9718) overnight at 4 °C. Samples were subsequently stained with AlexaFluor 488-conjugated goat anti-rabbit secondary antibody (1:400) for 1 h and analyzed on a FACS Calibur (Becton Dickinson). Data were analyzed with FlowJo software.