Anti sprouty2 60719

For immunoprecipitation total cell lysates (TCL) of U251 cells stably overexpressing Sprouty2-FLAG were prepared followed by sonication and centrifugation. Dynabeads™ M-280 Sheep anti-mouse IgG (Invitrogen, Carlsbad, CA, USA ) was conjugated with anti-FLAG (Cell signaling #8146, 1:50) overnight at 4°C. Protein lysates were incubated with anti-FLAG-conjugated beads for 1 h at 4°C. After three washes beads were boiled in loading buffer and analyzed together with TCL by immunoblotting (IB). TCL were prepared followed by sonication and boiling. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-FL-PVDF membrane (Millipore). Membranes were blocked with Odyssey^® blocking buffer (LI-COR Biosciences) in PBS and incubated with primary antibodies (Abcam: anti-Sprouty2 #60719, 1:500; Cell Signaling: anti-GAPDH #5174, 1:1,000; anti-tubulin #2128, 1:1,000; anti-vimentin #5741, 1:1,000; anti-FLAG #8146, 1:1,000). The secondary fluorescent-linked antibodies (IRDye^® 680RD goat anti-mouse and IRDye^® 800 CW goat anti-rabbit, 1:20,000; LI-COR Biosciences) were detected by the Odyssey FC Imaging System (LI-COR Biosciences).

Cells were fixed with 4% buffered formaldehyde solution (made from paraformaldehyde) for 15 min at RT, permeabilized with 0.5% Triton X-100 for 10 min and treated with 10% goat serum for 1 h at RT for blocking unspecific binding sites. Primary antibodies (Abcam: anti-Sprouty2 #60719, 1:160; Cell Signaling: anti-β-tubulin #2128, 1:100; anti-vimentin #5741, 1:100; anti-FLAG #8146, 1:1,600; anti-pERK #9101, 1:400; anti-Rab7 #9367, 1:100) were diluted in 0.3% BSA in phosphate buffered saline (PBS) and incubated overnight at 4°C. After three washings with PBS secondary antibodies (coupled to Alexa Fluor) were added in a dilution of 1:1,000 for 2 h at RT followed by washing and NucBlue/Dapi staining (Molecular Probes). No unspecific binding of the secondary antibodies was observed. At least three independent experiments with n > 100 cells were performed.