Secondary infrared dye antibodies

Embryos were dissected as previously described, and E9.5 dorsal telencephalon explants were isolated. Samples were lysed in lysis buffer (25 mM Tris at [pH 7.9], 150 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 10% glycerol, and 1 mM DTT) as well as 1X Proteinase Inhibitor Cocktail (Roche) and 1x PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Samples were then treated to at least cycles of snap freeze and thaw at −80ºC and stored at −80ºC until use. Protein was quantified with a Bio-Rad protein assay. Five to seven micrograms of protein was resolved by SDS-PAGE (Bio-Rad 4%–15% Mini-PROTEAN TGX Gel). Protein was then transferred to Immobilon-FL (Millipore), further processed by immunodetection, and blots scanned on a Li-Cor Odyssey Scanner. Antibody dilutions were Fgf15 1:200 (Santa Cruz; sc-16816) and GAPDH 1:1,000 (Santa Cruz; sc-365062). Secondary infrared-dye antibodies from Li-Cor used were diluted 1:15,000. Odyssey Software was used to quantify images.

The activation of caspase8 and the level of protein expression of Fas and FADD, were detected by immunoblotting. Briefly, 2×10⁵ cells were transfected with pEGFPC1 vector alone or with GFP-HGV-APT. The expression of GFP and GFP-HGV-APT was confirmed by fluorescence microscopy. At the indicated time periods green cells were sorted using BD FACSAria III cell sorter (Becton Dickinson, Palo Alto CA, USA), whole cell lysates were prepared from green fluorescent cells using RIPA Buffer and according to the manufacture's instructions (Thermo-Scientific). Proteins (30 μg) were separated by denaturing SDS-PAGE and then transferred onto a PVDF membrane. The membranes were blocked in 5% non-fat dry milk in TBS 0.1% Tween and then incubated overnight with the following primary antibodies: anti-active caspase8 (Cell Signaling Technology, Beverly, USA), anti FAS, and anti FADD antibodies (Santa Cruz Biotechnology, Inc.) at 4 °C. Then, the blots were incubated with the corresponding InfraRed dye secondary antibodies (Licor). The visualization of the membrane was carried using Licor membrane scanner.