Skip to main content
. 2018 Jul 18;7:e37993. doi: 10.7554/eLife.37993

Figure 2. Characterization of p18 in hippocampal neurons.

(A) Images of cultured hippocampal neurons co-immunostained for lysosomal protein LAMP2 (green) and p18 (red). Insets are enlarged images of LAMP2- and p18-immunoreactive puncta along the dendrites. Arrowheads indicate co-localized puncta. Scale bar: top, 20 μm; inset, 10 μm. (B) Left, p18 forms a complex with p14 and MP1 in hippocampal neurons. Lysates from cultured hippocampal neurons were immunoprecipitated with an anti-p18 antibody or control IgG and probed with the indicated antibodies. Right, RagA co-immunoprecipitates RagB, RagC, p18, and p14. Lysates from mouse hippocampi were immunoprecipitated with an anti-RagA antibody or control IgG and probed with the indicated antibodies. (C) Images of cultured hippocampal neurons co-immunostained for p18 (magenta) and LAMP2 (red). Neurons were infected with shRNA AAV directed against p18 with GFP co-expression or scrambled shRNA control before processing for immunofluorescence assay and imaging. Scale bar, 20 μm. (D) Images of hippocampal neurons stained for LAMTOR4 (red). Cells were infected and processed as in (C). Scale bar, 10 μm; inset, 5 μm. (E) Images of hippocampal neurons stained for RagA (red). Cells were infected and processed as in (C). Scale bar, 10 μm; inset, 5 μm. (F) Quantification of fluorescent signals for p18 (n = 13, p<0.001), LAMTOR4 (n = 11, p<0.001), RagA (n = 6, p=0.002), and LAMP2 (n = 6, p=0.356) in control shRNA and p18 shRNA-infected neurons shown in C– E. Student’s t-test. Note that n refers to the number of culture dishes analyzed. (G) Left, Western blot analysis of p18, p14, LAMTOR4, RagA, RagB, and LAMP2 in enriched lysosomal fractions prepared from WT neurons transfected with Accell control or p18 siRNA. Right, quantitative analysis of blots. Results are expressed as % of values in control siRNA-transfected WT neurons and shown as means ± SEM N = 3 independent experiments, p<0.001 for p18, p14, LAMTOR4, and RagA, p=0.001 for RagB (unpaired, two-tailed Student's t-test). See also Figure 2—figure supplement 1 and Figure 2—source data 1.

Figure 2—source data 1. Quantitative analyses of images and Western blots used for Figure 2 and Figure 2—figure supplement 1.
elife-37993-fig2-data1.xlsx (10.5KB, xlsx)
DOI: 10.7554/eLife.37993.007

Figure 2.

Figure 2—figure supplement 1. Localization of LAMTOR4 and RagA in hippocampal neurons, and representation of organelle marker proteins in individual OptiPrepTM fractions.

Figure 2—figure supplement 1.

(A) Images of cultured hippocampal neurons co-immunostained for lysosomal protein LAMP2 (green) and LAMTOR4 (red). Scale bar = 10 μm. (B) Images of hippocampal neurons co-immunostained for lysosomal protein LAMP2 (green) and RagA (red). Scale bar = 10 μm. (C) Three individual fractions collected after density gradient centrifugation of cultured neurons were assessed using antibodies against cathepsin B (lysosomes) and COXIV (mitochondria).