Expression of AZIN2 in exocrine glands.

<p>The sweat-producing acinar epithelium of a normal sweat gland shows strong AZIN2 expression while the ductal cells (upper right corner) are negative (a). Lobular and ductal epithelium in non-lactating breast shows a weak reactivity (b). Tear gland epithelium shows AZIN2 positivity (c) in a distribution similar to that seen for Rab27b (d).</p

Staining of normal gastric mucosa (corpus).

<p>AZIN2 reactivity in a tubular pattern in parietal cells (insert) (a). A consecutive section stained with mAb to H,K-ATPase revealed a similar distribution (b). Confocal microscopy of double immunofluorescence staining of AZIN2 (e) and H,K-ATPase (c) show co-distribution in the merged picture (d).</p

Expression of AZIN2 in megakaryocytes in normal bone marrow.

<p>Insert: A megakaryocyte at higher magnification showing a granular cytoplasmic staining.</p

Expression of AZIN2 in neural tissue.

<p>Nerve cells in a spinal ganglion stained with K3 (a) and control staining with pre-immunization serum from the same rabbit (b). <i>In situ</i> hybridization with sense RNA probe (c) and anti-sense (control) probe (d). Staining of a portion of <i>Nucleus dentatus</i> (e) and of the granular cell layer in cerebellum (f).</p

Upregulation and redistribution of AZIN2 in MCs during activation.

<p>RBL-1 (A) and HMC-1 (B) cells were activated with PMA and A23187 for the times indicated. After activation, the cells were either immunostained for AZIN2 (antiserum 2) or harvested for quantitative real-time PCR analysis of AZIN2. Relative mRNA levels are expressed as the ratio of AZIN2 mRNA level to that of either rat β-2-microglobulin (β2 m) or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Original magnification of all immunostainings: ×400.</p

Polyamine depletion inhibits serotonin release from MCs.

<p>LAD2 (A, C) and primary human MCs (B, D) were cultured in polyamine-free medium and sensitized overnight with human myeloma IgE. Before activation, the cells were and treated with 2 mM DFMO as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006858#s2" target="_blank">Materials and Methods</a>, and subsequently activated with anti-IgE for the indicated times; and the conditioned medium was analyzed for serotonin (A–B) and β-hex (C–D) release. Symbols represent treatment of MCs: anti-IgE only (□), anti-IgE + 2 mM DFMO + 0.5 mM putrescine (▪), anti-IgE + 2 mM DFMO (○), non-stimulated (•).</p

Immunohistochemical identification of AZIN2 protein in human cutaneous mastocytosis.

<p>Human skin tissue sections stained histochemically with either antiserum 3 to AZIN2 (A) or rabbit preimmune serum (B). Original magnifications: ×400 (A–B) and ×1000 (inset). Note the cytoplasmic granular-like staining pattern of AZIN2 in the inset.</p

Specificity testing of AZIN2 antisera.

<p>(A) Immunoblot from COS-7 cells transfected with plasmids carrying FLAG-tagged AZIN1, AZIN2, or ODC gene or an empty FLAG-vector (Ctrl). An Odyssey Infrared Imager was used for simultaneous two-color detection of rabbit anti-AZIN2 (antiserum 3) and mouse anti-FLAG antibodies. The lowest panel represents merged blots, yellow indicating co-localization of anti-AZIN2 and anti-FLAG. (B) Confocal laser scanning microscopy of COS-7 cells expressing one of the following genes: AZIN2-FLAG, AZIN1-FLAG, or ODC-FLAG. The cells were stained with either AZIN2 antiserum 2 or 3 together with mouse anti-FLAG, followed by donkey anti-rabbit Alexa Fluor-555 and donkey anti-mouse Alexa Fluor-488. AZIN2-FLAG-transfected cells were also stained with preimmune sera of the corresponding rabbits. In the merged images, yellow indicates the co-localization. Bars represent 10 µm.</p