In silico analysis of rat NECC2 sequence.

<p><i>A</i>. Schematic representation of the structural and functional motifs predicted in rat NECC2 amino acid sequence. <i>B</i>. Schematic representation of the genomic structure of rat <i>Necc2</i> coding for the <i>Necc2</i> isoform containing the HR domain and a newly identified transcript lacking this domain (<i>Necc2</i>α). Arrows indicate the location of the paired primers used to amplify both <i>Necc2</i> transcripts. <i>C</i>. Standard PCR amplification in PC12 cells shows two PCR products with the expected sizes for the two rat <i>Necc2</i> transcripts (left panel; primers a and b). Nested PCR amplification of <i>Necc2</i> transcript (central panel; primer c) or the <i>Necc2</i>α transcript (rightmost panel; primer d) using specific internal reverse primers. Non-DNA samples (C-) are shown as controls for exogenous contamination.</p

NECC2 associates to caveolae both as an integral membrane protein and a peripheral membrane protein.

<p><i>A</i>. Immunoblot analysis of whole cell lysates from PC12 cells with the anti-NECC2 antibody. As control, anti-NECC2 antibody was pre-incubated with an excess of antigen. <i>B</i>. Cytosolic (S) and crude membrane (P) fractions from PC12 cells were obtained by subcellular fractionation as described in Methods and subsequent analyzed by immunoblotting. As shown by anti-NECC2 antibody immunolabeling, NECC2 distributes in the cytosol and, to a lesser extent, it also associates with membrane fractions. In contrast, exogenous full-length cMyc-NECC2 distributed to both fractions, with a higher content in the membrane fraction. The distribution of EEA1, TrkA, GM130, actin and caveolin-1 was also analyzed. <i>C</i>. Caveolae-enriched membranes from PC12 cells were isolated by using a detergent-free method based in a discontinuous sucrose gradient (5-35-45%). Distribution of endogenous NECC2 and cMyc-tagged NECC2 variants were assayed by immunoblot. Neither NECC2 nor NECC2ΔHR co-migrated with caveolin-1 and TrkA to the buoyant fraction (fraction 2).</p

Overexpression of NECC2 inhibits NGF-mediated TrkA signaling pathway.

<p><i>A</i>. Representative confocal images of PC12 cells transfected with cMyc-<i>Necc2</i>ΔHR and double-stained with anti-cMyc and anti-TrkA antibodies. Immunofluorescent signals significantly overlap at the cell periphery and intracellularly as shown in the binary mask (right panels). Scale bars, 10 µm. <i>B</i>, <i>C</i>, <i>D</i> and <i>E</i>. PC12 cells transiently transfected with full-length cMyc-<i>Necc2</i>, cMyc-<i>Necc2</i>ΔHR, or the empty vector (mock) were grown to 90% confluence and exposed for 4 h to serum-low differentiation media before NGF stimulation for the indicated time points. Whole cell protein extracts were then subjected to immunoblot with Akt and phospho-Akt (pAkt) antibodies (<i>B</i> and <i>C</i>) or with ERK and phosphor-ERK (pERK) antibodies (<i>D</i> and <i>E</i>). Quantitative data were represented as ratio of pAkt <i>vs</i>. Akt or pERK vs. ERK, respectively. The data represent the means (± SEM) of three independent experiments. <i>P</i> < 0.05 <i>vs</i>. corresponding control (unpaired, 2-tailed t test).</p