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. 2016 Feb 1;2:16004. doi: 10.1038/cddiscovery.2016.4

Figure 6.

Figure 6

NGF induces TrkA/cFLIP binding and reduces the level of cFLIP at death receptors. (a) Representative co-immunoprecipitation western blots demonstrating endogenous cFLIP pull-down of TrkA in immunoprecipitates from total cell extracts (1 mg) of NGF-treated (100 ng/ml for 6 h) but not untreated TrkA SH-SY5Y cells. (b) Representative co-immunoprecipitation western blots demonstrating exogenous FLAG-tagged cFLIP pull-down of TrkA by immunoprecipitates in whole cell extracts (300 μg) of NGF-treated (100 ng/ml for 1 and 6 h) but not untreated TrkA SH-SY5Y cells transiently transfected with a FLAG-tagged cFLIP expression vector. Note the reduced levels of TrkA pulled down by exogenous FLAG-tagged cFLIP in TrkA SH-SY5Y cells co-treated with CEP-701 (100 nM) and NGF for 6 h. Input levels of TrkA and cFLIP are also provided (Input). (c) Representative western blots demonstrating reduced levels of cFLIP and increased levels of caspase-8 in TRAIL-activated DR4-positive death receptor complexes purified from TrkA SH-SY5Y cells preincubated for 6 h with NGF (100 ng/ml) and then treated for 1 h with biotinylated-TRAIL (500 ng/ml) (NGF TRAIL) as compared with TrkA SH-SY5Y cells preincubated for 6 h in the absence of NGF then treated for 1 h with biotinylated-TRAIL (500 ng/ml) (TRAIL) and TrkA-SH-SY5Y cells treated for 6 h with NGF (100 ng/ml) alone (NGF), and untreated TrkA SH-SY5Y cells (Con). Input levels of caspase-8, cFLIP and α-tubulin are provided (Input).