Anti mouse igg cy3

A549 and H1299 cells (classified as NSCLC cells) were collected from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium enriched with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin sulfate. The possible mycoplasma contamination was checked by Mycotest kit (Invitrogen, Chicago, IL, USA). The following antibodies were used in the study: pATM, FANCD2, Chk1, β-Actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); Rad18 from Bethyl Laboratories, Inc. (Montgomery, TX, USA); pATM-Ser1981, pChk1-Ser317, pChk2, FANCJ, Bid, caspase-3, Snail and cyclin B are from Cell Signaling Technologies (Danvers, MA, USA); and γH2AX from Millipore company (Darmstadt, Germany); and E-cadherin from Becton Dickinson company (BD Biosciences, San Jose, CA, USA) Cell Signaling Technologies (The horseradish peroxidase (HRP)-conjugated secondary antibodies, such as anti-mouse IgG-Cy3 and anti-rabbit IgG, were procured from Molecular Probes (Santa Cruz, CA, USA).

The NSCLC cell lines A549 (human adenocarcinoma epithelial cell line) and H1299 (human were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% FBS, 100 μg/ml streptomycin sulfate and 100 U/ml penicillin. Normal human bronchial epithelial cells were grown in BEGM™ Bronchial Epithelial Cell Growth Medium as described previously [51 (link)]. Cells were routinely tested for mycoplasma contamination using Mycotest kit (Invitrogen) and cells within 10 passages were used in the experiments. AITC and PITC (Sigma, St. Louis, MO) stock solutions were prepared by dissolving in anhydrous DMSO and stored at −20°C. These stock solutions were further diluted to required concentration before adding to the cells. Antibodies to the following antigens used in this study include: ATR, ATM, Chk1, FANCD2 and GAPDH were from Santa Cruz Biotechnology, Inc.; Rad18, from Bethyl Laboratories, Inc.; phospho-ATM-Ser1981, phospho-Chk1 Ser-317 were Cell Signaling Technologies, γH2AX is from Millipore. The secondary antibodies like anti-mouse IgG-Cy3, anti-mouse IgG-FITC, anti-rabbit IgG-FITC were from Molecular Probes.

Cells were washed with PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich), and permeabilized with 0.1% Triton X-100 for 10 min. Fixed samples were blocked with 3% skim milk in PBS for 30 min and then incubated with primary antibody diluted in 1% skim milk in PBS for 1 h. After washing with PBS, cells were incubated with anti-mouse IgG-Cy3 (Molecular Probes, Eugene, OR) then 1 μg/ml Hoechst 33342 was used for DNA staining. Immunofluorescence was observed with an Olympus upright fluorescence microscope (BX50F).