Alexa fluor goat anti rabbit igg h l

Cells were transduced with viral sensor sgRNAs (sgRNAs are in Table 2). 2 d after transduction, cells were subjected to a second transduction with SETDB1-specific sgRNAs or an NTC control sgRNA. Cells were selected with 1 µg/ml puromycin (viral sensor sgRNAs) and 5 µg/ml blasticidin (SETDB1 or NTC sgRNAs). Cells were harvested for FACS analysis 14 d after the second transduction. In brief, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then washed with 1 ml PBS. Cells were permeabilized with PBS + Tween 0.1% for 15 min. Primary antibodies were used at a 1:100 dilution (MAVS; ab31334; Abcam; MDA5; ab69983; Abcam) in 150 µl FACS buffer (PBS with 0.5% BSA and 2 mM EDTA) at 4°C for 30 min. The cells were washed twice with 1 ml FACS buffer. The secondary was Alexa Fluor goat anti–rabbit IgG (H + L; A11008; ThermoFisher) and was used at a 1:500 dilution in 150 µl FACS buffer. Cells were washed twice with 1 ml FACS buffer and analyzed on a LSRII analyzer (BD).

HeLa cells were cultured in Dulbeco’s modified eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were synchronized using a sequential thymidine (2.5 mM; Sigma-Aldrich) and aphidicolin (5 μg/mL; Tocris Bioscience) block-and-release protocol. Stock solutions of SR-1227 and SR-3029 were prepared in DMSO; a corresponding volume of DMSO was used for all negative controls.
For microscopy, cells were seeded onto 25 mm coverslips that were contained in six-well plates. Cells were washed once with cold PBS and once with 100% cold methanol, followed by fixation with 100% cold methanol for 15 min at −20°C. Cells were then washed 3x with PBS + 0.1% Tween-20 at RT, followed by blocking with 2% normal goat serum in 0.1% Triton-X100 in PBS for 10 min. Cells were incubated with rabbit anti-gH2AX (Cell Signaling Technology, 2577S), then DRAQ5 (Thermo Fisher Scientific) or DAPI and secondary antibodies (Alexa Fluor Goat-anti Rabbit IgG (H+L), ThermoFisher; 1:500) for 45 minutes at RT. Coverslips were mounted on slides using Prolong Gold antifade mounting media.
For western blotting, ~40 μg whole cell lysate was incubated with antibodies against γ-tubulin (Sigma-Aldrich, GTU88), phospho-Chk1 (Cell Signaling Technology, 2348S), or phospho-Histone H3 (Sigma, H0412).