<i>EVER1</i> and <i>EVER2</i> are expressed in lymphocytes.

<p><b>A.</b> Expression of <i>EVER1</i> and <i>EVER2</i> genes in different mouse organs was assessed by qRT-PCR. Expression in the spleen was set as 100%. For each organ at least 3 independent experiments were performed. <b>B.</b> Expression of <i>EVER1</i> and <i>EVER2</i> genes in murine T and B cells was determined by qRT-PCR. At least 3 independent experiments were performed for each cell type. Expression of EVER1 protein in murine splenocytes (<b>C</b>) and purified T cells (<b>D</b>) was determined by western blot. An EVER1-specific antibody (Abcam; ab67326) was used. A single band at ≈100 kDa was detected (predicted EVER1 MW = 91 kDa). <b>E.</b> 293 T cells were transfected with plasmids encoding either EVER1-FLAG or EVER2-FLAG fusion protein. The fusion protein was immunoprecipitated with anti-FLAG antibody and subsequently a western-blot was performed using the anti-FLAG antibody or two different anti-EVER1 antibodies (Ab n^o1 - Abcam; ab67326; Ab n°2 - Osenses; OSR00223W).</p

Zinc homeostasis in T cells.

<p>Purified murine naive CD8+ (A) or CD4+ T cells (B) were activated with immobilized anti-CD3 and anti-CD28 Abs and expression of the <i>ZIP10</i> zinc transporter and <i>EVER1</i> genes was determined by qRT-PCR at the indicated time points. Data are from 3 independent experiments. Total amount of free zinc was determined in murine CD8+ (<b>C</b>) or CD4+ T cells (<b>D</b>) after 24 h incubation in standard medium (medium), in medium supplemented with IL-2 (IL-2), or with anti-CD3/anti-CD28 Abs in the presence of IL-2 (CD3/CD28). The content of free zinc was measured by flow cytometry with FluoZin-3, a zinc ion-specific indicator. The total amount of free zinc per cell was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039995#s3" target="_blank">materials and methods</a> and normalized for the unstimulated cells (medium) values (100%). Purified naive CD8+ (<b>E</b>) or CD4+ T cells (<b>F</b>) were stimulated by immobilized anti-CD3 and anti-CD28 Abs in the presence of IL-2 and IL-12. The expression of <i>metallothionein 2</i> (MT-2) was assessed by qRT-PCR. Data are representative of 3 independent experiments. <b>G</b>. Murine naive CD8+ or CD4+ T cells were activated (CD3/CD28) or not (control) by immobilized anti-CD3 and anti-CD28 Abs for 24 h The concentration of free zinc was then measured by flow cytometry with FluoZin-3. <b>H</b>. Purified murine naive CD8+ T cells were activated with immobilized anti-CD3 and anti-CD28 Abs. At the indicated time the zinc ionophore pyrithione (PY) was added to the medium at different concentrations. Radiolabeled thymidine was added 24 hrs after initiation of the cell culture, and proliferation was assessed on the basis of the thymidine incorporation 24 h later.</p