Els of phosphorylated Smad2 (P-Smad) in lysates from primary mouse neurons. Because these cultures have high basal endogenous levels of TGF- signaling, we were able to measure Smad phosphorylation in the absence of exogenous TGF-. Knockdown of beclin 1 resulted in a significant decrease in the ratio of phosphorylated to total Smad2 (to 26 of control levels, Fig. 7a, b). Total levels of Smad2/3, however, were not changed (Fig. 7c). To further corroborate these data using a different method, we took advantage of a TGF- responsive reporter cell line. F11 cells are fibroblasts isolated from TGF-1-/- mice that have been engineered to stably express secreted alkaline phosphatase (SEAP) underaGFPInfected CellsHARecycled ALKALKTotal ALKbRecycled/Total ALK5 (Integrated Density/cell)cbec shRNA15 10 5 0 -ctrl shRNA***ctrl shRNAbec shRNAFig. 6 Beclin 1 regulates ALK5 recycling in COS7 cells. Representative images of COS7 cells transiently expressing HA-ALK5 and infected with (a) ctrl or (b) bec shRNA lentivirus. Only infected (GFP+) cells were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 analyzed. Recycled ALK5 was detected using a mouse anti-HA antibody and a goat-anti-mouse Alexa 647 secondary antibody. Total ALK5 was detected using a rat-anti-ALK5 and goat-anti-rat Alexa 555. c Combined results from three independent experiments. Quantification of recycled/total ALK5 from confocal z-stacks. Data is expressed as the ratio of recycled/total ALK5 summed over the z-stack for each cell where each dot represents one cell (n = 30 fields/group, n > 85 cells/group). ***p 0.001. Bar graphs are mean ?SEMO'Brien et al. Molecular Neurodegeneration (2015) 10:Page 9 ofcontrol of the SBE promoter [32]. Treatment of these cells with exogenous TGF- induces SEAP secretion into the media, which can be collected and assayed for activity. Infection of F11 cells with beclin 1 shRNA lentivirus resulted in a significant decrease in SEAP activity compared to cells infected with the control virus (Fig. 7e). This supports our P-Smad data and suggests that beclin 1 regulation of TGF- signaling is conserved among cell types. We also used this method to test the specificity of the effect of beclin 1 knockdown on TGF- signaling. Beclin 1 is a subunit of the core type III PI3K complex containing the kinase VPS34 and the activating subunits VPS15 and Ambra (Fig. 7d). Beclin 1 also binds ATG14 and UVRAG (a homolog of yeast Vps38) in a mutually exclusive manner. The yeast homologs of these complexes regulate autophagy and protein sorting, respectively [30, 31, 33, 34]. To determine if regulation of TGF- signaling is specific to beclin 1 and to distinguish between these beclin 1 complexes, we targeted various components of the PI3K complexes and the autophagy pathway. F11 cells were transfected with plasmids encoding shRNA against VPS34, UVRAG, ATG14 or a control shRNA (Fig. 7e). Knockdown ofeither VPS34 or UVRAG impaired SEAP activity. However, knockdown of ATG14 did not affect SEAP activity. Likewise, knockdown of ATG7, another regulator of autophagy that does not bind beclin 1, had no effect on SEAP activity. Taken together, these results suggest that TGF- signaling requires a complex of beclin 1, the PI3K VPS34, and UVRAG. These data are consistent Capecitabine with the protein sorting function of this complex in yeast, and indicate that regulation of TGF- signaling is dependent on beclin 1, but independent of its role in autophagy.Discussion Despite the importance of TGF- signaling in neuronal development and homeostasis, little i.