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

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

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

NECC2 expression and distribution are regulated by NGF.

<p><i>A</i>. Representative confocal images of HA-<i>TrkA</i> transfected PC12 cells under basal conditions or treated with NGF at the indicated time points. After treatment, cells were subjected to double-immunofluorescence using anti-NECC2 and anti-HA antibodies. The colocalization channel was isolated using Imaris 6.4 (Bitplane) and shown alone in the images on the far bottom. Mander’s coefficient A (between NECC2 and TkA) was calculated to quantify the degree of colocalization and represented as the mean ±SEM of at least 5 cells per experimental group. a, <i>P</i> < 0.05 <i>vs</i>. control; b, <i>P</i> < 0.05 <i>vs</i>. 5 min; c, <i>P</i> < 0.05 <i>vs</i>. 30 min (unpaired, 2-tailed t test). <i>B</i>. Representative micrographs of PC12 cells immunolabeled with anti-NECC2 during long-term (1-4 days) stimulation with NGF. NECC2 staining localizes beneath the plasma membrane as well as in puncta/vesicular-like structures in growing neurites and tips. <i>C</i>. Double-immunostaining of NGF-differentiated PC12 cells with anti-NECC2 and anti-caveolin-1 sera. Scale bars, 10 µm. <i>D</i>. Protein extracts from NGF-stimulated PC12 cells during differentiation (1-4 days) were analyzed by immunoblotting using the anti-NECC2 antibody. Quantitative data were represented as ratio of NECC2 vs. Ponceau. The data represent the mean (± SEM) of three independent experiments. a, <i>P</i> < 0.05 <i>vs</i>. corresponding control; b, <i>P</i> < 0.05 <i>vs</i>. 1- or 2-days treated cells (one-way ANOVA followed by <i>Newman–Keuls</i> test).</p

Intracellular distribution of endogenous NECC2 in PC12 cells.

<p><i>A</i>. Representative confocal images of a PC12 cell immunolabeled with the anti-NECC2 antiserum. NECC2 distributes throughout the cytoplasm and in close apposition to the cell membrane. Specificity of the signal was tested by preadsorption of the anti-NECC2 antibody with excess of antigen. <i>B</i>. PC12 cells were double-stained with antibodies against NECC2 (red) and caveolin-1 or actin (green) (top and bottom panels respectively). Significant overlap between markers at the cell periphery is shown in the binary mask at the rightmost panels. <i>C</i>. Prior to double immunostaining with anti-NECC2 and anti-caveolin-1, PC12 cells were treated with 5 µmol/L of LatB for 30 min at 37°C. Scale bars, 10 µm.</p

Analysis of the intracellular localization of cMyc-tagged NECC2 variants.

<p><i>A</i>. Schematic representation of NECC2 constructs and its truncated variants lacking the hydrophobic region (NECC2ΔHR) or containing the first 4 N-terminal coiled-coil regions (NECC2Δ372). <i>B</i>. Confocal images of PC12 cells co-expressing cMyc-NECC2 or its truncated forms and CFP-caveolin-1 (CFP-cav1). Full-length cMyc-NECC2 accumulates in the perinuclear area. NECC2ΔHR and NECC2Δ372 immunosignals accumulate intracellularly and in the proximity of the cell surface. NECC2 immunofluorescence signal strongly overlaps with CFP-caveolin-1 as shown in the binary mask (rightmost panel). Scale bars, 10 µm.</p

Reduction of endogenous NECC2 by RNA interference impairs NGF-mediated TrkA signaling.

<p><i>A</i>. PC12 cells were stably transfected with pEGFP-shRNA or <i>Necc2</i>-shRNA plasmid (siRNA) and whole protein extracts were analyzed for immunoblotting using anti-NECC2 antibody and anti-actin antibody. <i>B</i> and C. PC12 cells overexpressing pEGFP-RNAi (MOCK) or <i>Necc2</i>-shRNA (R1) were grown to 90% confluence and exposed for 4 h to serum-low differentiation media before NGF stimulation for the indicated time points. Cell lysates were subjected to immunoblot with Akt and phospho-Akt (pAkt) antibodies (<i>B</i>) or with ERK and phospho-ERK (pERK) antibodies (<i>C</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