Tained from American Kind Culture Collection (ATCC) (MCF7-ATCC). The MCF7-Syk cell line was established by stably reconstituting MCF7-BD cells with Syk-EGFP. MDA-MB-231 breast cancer cells have been obtained from ATCC. A line of MDA-MB-231 cells expressing SykEGFP in response to tetracycline were described previously [26]. All cell lines have been cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 7.5 FBS, 100 U/ml penicillin and 100 /ml streptomycin. 2.two. Antibodies and also other reagents Major antibodies against Syk (N19), Myc (9E10), HA (Y-11), calpain four (capn4) (P-1), and RelA (F-6) have been obtained from Santa Cruz Biotechnology. Anti-CAST (for Western blotting), anti-calpain 1 (capn1), and anti-PARP antibodies had been from Cell Signaling Technology. Anti-GAPDH antibody was from Ambion. Anti-phosphotyrosine antibody (4G10), anti-CAST (for immunostaining), mouse anti-human CD29 (integrin 1), and protein A-Sepharose beads covalently conjugated with 4G10 for immunoprecipitation were purchased from Millipore. Monoclonal anti-FLAG M2 antibody was purchased from SigmaAldrich. GFP-Trap-A beads for immunoprecipitation of GFP-fusion proteins have been purchased from Chromotek. Each goat anti-rabbit and goat anti-mouse horseradish peroxidase-conjugated secondary antibodies for Western blotting were from Thermo Fischer Scientific. Alexa Flour 594 labeled goat anti-mouse IgG for immunostaining was obtained from Invitrogen.1377584-27-4 Order Calpeptin and staurosporine have been purchased from Sigma-Aldrich.Formula of 1211526-53-2 Calpain 1 purified from human erythrocytes as well as the recombinant CAST peptide were obtained from Calbiochem.PMID:26780211 t-Butoxycarbonyl-leucine-methionine-7-amino-4-chloromethylcoumarin (tBoc-Leu-Met-CMAC) was purchased from Invitrogen. Recombinant human TNF- was purchased from R D Systems. Calpain-Glo Protease Assay and Dual-Luciferase Reporter Assay Systems were from Promega. ProteSEEKER protease inhibitor kit was obtained from G-Biosciences. Rhod-3 imaging kit was bought from Invitrogen. 2.three. Immunoprecipitation For immunoprecipitation experiments, cells had been lysed in buffer containing 50 mM Tris/ HCl, pH 7.five, 150 mM NaCl, ten glycerol, 1 NP-40, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM Na3VO4, and 0.two (v/v) protease inhibitor cocktail. Pre-cleared lysate was incubated with protein G-Sepharose beads pre-mixed with principal antibody and agitated at 4 for 4 h. The immune complexes have been washed four times with lysis buffer and resuspended in SDS sample buffer. In some experiments, cells were treated with sodium orthovanadate (50 ) mixed with H2O2 (50 mM) to generate pervanadate (PV), which was added to the culture medium for a 15 min incubation on ice just just before cells had been harvested. Proteins have been resolved by SDS-PAGE and detected by Western blotting making use of theNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptBiochim Biophys Acta. Author manuscript; offered in PMC 2014 October 01.Fei et al.Pageappropriate key antibody and horseradish peroxidase-conjugated secondary antibody making use of enzyme-linked chemiluminescence (ECL) reagents (PerkinElmer).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript2.four. Immunofluorescence Cells seeded on coverslips were fixed in 3.7 formaldehyde, permeablized and blocked in phosphate-buffered saline (PBS) containing 0.five Triton X-100 and five goat serum and stained with anti-CAST major antibody. Bound antibody was detected with Alexa Fluor?594 goat anti-mouse IgG (1:1000). 4′,6′-Diamidino-2-phen.