Schematic representation of the antisera library generation.

<p>Schematic representation of the antisera library generation.</p

FACS analysis with sera specific for well-known proteins.

<p>(A) Comparison of the CD8 staining performed on PBL with either a commercially available anti CD8 mAb (BD biosciences) or the anti CD8 alpha antiserum at 1∶100 dilution points. Both the samples were stained also with commercially available anti CD3 and anti CD4 mAb (BD biosciences). The distribution of CD4 and CD8 is analyzed upon gating on CD3 positive cells. (B) Examples of staining with antisera from the library. PBLs from healthy donors were stained with anti CD2, CD1d, CD8 alpha, CD25, CD72, CD80, CD38, and CD86. The expression of CD25 was assessed upon a 24 hours activation of PBLs with 1 µg/ml of PHA. The expression of CD80 and CD86 was assessed upon gating on monocytes after a 24 hours activation of PBLs with 1 µg/ml of PHA. The expression of CD133 was analyzed on cord blood derived CD34+, CD45dim cells. Serum from not immunized mice was used as negative control in all the stainings.</p

Comparison of the results obtained with antisera specific for well-known proteins assessed both by FACS on PBMCs from healthy donors and by IHC on inflamed lymph nodes.

<p>Comparison of the results obtained with antisera specific for well-known proteins assessed both by FACS on PBMCs from healthy donors and by IHC on inflamed lymph nodes.</p

Results of sera screening by FACS on PBL and Cord Blood cells.

<p>(A) FACS analysis of sera positive on PBLs. PBLs were stained with the indicated sera at the optimal dilution point (1∶50 to 1∶200). The samples were stained also with anti CD3, anti CD19 and anti CD56 mAbs to analyze the sera reactivity upon gating on the different subpopulations. A plot representative of five different donors is shown for each serum. (B) KRTCAP-3 specific serum recognizes PHA-treated cells. PBMCs are treated for 24 hours with 1 µg/ml of PHA. After the treatment both un-stimulated and treated cells are stained with the KRTCAP-3-specific serum. (C) FACS analysis of sera positive on cord blood cells. Cord blood mononuclear cells are stained with the indicated sera at the optimal concentration (1∶50 to 1∶100). The samples are stained also with anti CD45 and anti CD34 mAbs to perform the analysis upon gating on CD34highCD45dim. A plot representative of a least 3 independent donors is shown. Il all the cases (A,B,C,) a staining with the serum of not immunized mice was used as negative control. (D) RT-PCR analysis. a- cDNA from total PBMC were amplified with primers specific for the indicated proteins. b- cDNA from both un-stimulated and PHA-treated PBMC was amplified with KRTCAP-3 specific primers. KRTCAP3 expression is up regulated two to three times. Beta actin amplification is used as normalization. c- cDNA samples from CD34+CD45dim cells were generated by retro-transcription of RNA extracted from a pool of CD34 positive cells from 2–3 independent cord blood units magnetically purified using the Miltenyi CD34 microbeads kit according the manufacturer instruction. The purity of the CD34+CD45dim cells was usually >99%. The samples were amplified with primers specific for the indicated proteins and described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034395#s2" target="_blank">Methods</a> section.</p

Assessment of antisera specificity on Hela transfected cells.

<p>Hela cells were transiently transfected with a myc-tag version of the proteins identified with the sera library. At 24 hours from the transfection cells were lysated as described in the Method section. 40 µg of total proteins were loaded on SDS page and a WB analysis was performed using both an anti myc mAb (9E10 clone) and the corresponding antiserum. (A) WB analysis of Hela cells transfected with CRISP-1 and MOSC-1. In both the cases the anti myc mAb and the specific antiserum recognized a protein of the expected molecular weight that is not present in the cells transfected with the mock vector. A comparable result was obtained wit KRTCAP-3 (B), TMCC-1 (C), TMEM38B (D) and SUSD3 (E) transfected cells. The WB analysis of GSG1-L cells (E) and LPPR2 cells (F) shows that neither the anti myc nor the specific antiserum is able to recognize in a specific way a protein in transfected cells.</p