Two hours before harvesting, 100 ul of 4 uM BD Calcein AM was added to washed cells. Plates were www.selleckchem.com/products/Vandetanib.html read at 485 nm and relative fluorescence units recorded. RFU of ten replicate wells were averaged and analyzed for significance. Mann Whitney unit analysis test was applied to relative fluorescent units data from 10 replicate wells and p values are reported. mouse anti RACK1 antibody . and rabbit anti RACK1 and anti Cbp/PAG antibodies. Mouse anti Lyn, clone 10A6. 2, and MilliplexW assays were from Millipore. Horse radish Inhibitors,Modulators,Libraries peroxidase conjugated secondary anti bodies were goat anti rabbit Ig and goat anti mouse Ig antibodies, anti rabbit light chain TrueBlotWantibodies and anti rabbit light chain TrueBlot IP beadsW. Cell lysates Inhibitors or equal volumes of DMSO solvent vehicle were added to adherent, serum starved cells in 6 well plates before preparation of cell lysates.
Where indi cated, cells were stimulated with 500 or 100 ng/ml of human EGF for five ten minutes at 37 C before medium was removed, and chilled cell lysis buffer imme diately added. Dissolving cells were sonicated 15 seconds before microcentrifugation for 20 minutes. Supernatants were removed and protein concentrations quantitated Inhibitors,Modulators,Libraries using Bio Rad Bradford protein assay.Generally 20 30 ug of protein were loaded into 7. 5% Tris HCl pre cast SDS PAGE gels. MILLIPLEXW MAP 8 Plex phospho Src family kinase immunoassay Quantitative sandwich immunobead assays were used to identify Y 419 phosphorylated SFK mem bers including Src, Yes, Fyn, Fgr, Lck, Hck, Blk and Lyn.
Inhibitors,Modulators,Libraries Cell free lysates of unstimulated NSCLC Inhibitors,Modulators,Libraries cell lines were incubated with specific antibody conjugated beads which select a SFK member, followed by addition of biotiny lated pan anti phospho Src to quantify the level of Y 419 phosphorylation of that SFK Inhibitors,Modulators,Libraries inhibitor order us member. Samples were read in a luminex 100 reader after addition of PE conjugated StrepAvidin. All assays were performed and analyzed with respect to a standard curve of Hela or Ramos cell lysates according to manufacturer recom mended protocols. Western blotting SDS PAGE were performed using pre cast 7. 5% Tris HCl gel and electrophoresed in Tris Glycine SDS buffer at 100 volts for 99 minutes. Sepa rated proteins at 20 30 ug/lane were transferred to PVDF membranes using a semi dry transfer apparatus. Blotted membranes were washed, blocked overnight on a rocker at 4 C, then incubated with 1 1000 primary antibody diluted in SignalBoost, 5% BSA, or 5% milk in TBST. Secondary antibodies were added at 1 2000 for 2 hours at 25 C. ECL substrate was added, then blots exposed to film before developing.