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Ed a significant decrease in ALK5 levels upon beclin 1 knockdown by confocal microscopy (Fig. 3b), which we confirmed by western blot (Fig. 3c, d). However, beclin 1 knockdown had no effect on levels of ALK5 mRNA (Fig. 3f). Although we were unable to detect the type II TGF- receptor TBRII by immunofluorescence, we measured the effect of beclin 1 knockdown on TBRII protein and mRNA levels. Like ALK5, TBRII protein (Fig. 3c, e) but not mRNA (Fig. 3g) levels are decreased by beclin 1 knockdown in primary neurons. These data indicate beclin 1 post-transcriptionally regulates levels of both TGF- receptors.Beclin 1 regulates ALK5 localization with recycling machineryOne reason for a decrease in receptor levels could be a failure to recycle these receptors back to the cell surface and their subsequent degradation. To determine if beclin 1 regulates localization of ALK5 in recycling endosomes, we examined the colocalization of ALK5 with a marker of recycling endosomes, Rab11. Rab11 has previouslyFig. 3 Beclin 1 knockdown reduces ALK5 protein levels. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 a Representative image of CF1 hippocampal neurons stained for beclin 1 and ALK5. Intensity trace (offset white line) is plotted at right. b Quantification of ALK5 fluorescence intensity in CF1 hippocampal cells infected with ctrl or beclin 1 shRNA. Only GFP+ infected cells were analyzed. Data are combined results from two independent experiments. Each dot represents a single cell (n = 37?1 total cells from 20 different fields/group). Data were analyzed by unpaired Student's t-test, ***p 0.001. c Representative western blot of lysates from infected CF1 forebrain neurons. d Quantification of ALK5 and e TBRII protein levels normalized to NSE. Data are combined results of three independent experiments analyzed by unpaired t-test. *p 0.05. f ALK5 and g TBRII mRNA levels were measured Capecitabine by RT-PCR and levels were normalized to GAPDH. Relative levels were calculated using the Ct method. Data are combined results from three independent experiments. Data were analyzed by unpaired Student's t-test. All bar graphs are mean ?SEM.O'Brien et al. Molecular Neurodegeneration (2015) 10:Page 6 ofbeen shown to be required for recycling of the TGF- receptors [25]. Beclin 1 knockdown decreased the colocalization of ALK5 with Rab11 (Fig. 4). In both yeast [31] and microglia [5], beclin 1 mediates receptor recycling by recruiting the retromer, a multiprotein complex composed of VPS35, VPS29, and VPS26, as well as a sorting nexin subcomplex. To determine if beclin 1 recruits the retromer to ALK5, we nextanalyzed colocalization between ALK5 and the retromer component VPS35. Knockdown of beclin 1 significantly decreased the fraction of ALK5 colocalizing with VPS35 (Fig. 5). These data together suggest that localization of ALK5 with the retromer is in part beclin 1-dependent and that in the absence of beclin 1, sorting of ALK5 into Rab11+ recycling endosomes is impaired. Taken together with the effect on ALK5 levels, these results area ALKctrl shRNArabmerge1 2 ALK5 rabFluorescence Intensity300 200 100 0 0 5 10distance ( )b ALKbec shRNArabmergeFluorescence Intensity150 100 50ALK5 rab2distance ( )cColoc coefficient0.***0.0.0.ctrl shRNAbec shRNAFig. 4 Localization of ALK5 in Rab11+ recycling endosomes is beclin 1-dependent. Representative images of neurons infected with (a) ctrl shRNA or (b) bec shRNA and stained with antibodies against ALK5 (pseudocolored green) and Rab11 (pseudocolored red). Regions in the box were brightened t.