Fig 2: Western blot analysis and measurement of CD45 and CD45RA and CD45RO activities. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 µg/ml each) mAbs for various periods of time, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using an anti-CD45 mAb and the chemiluminescence technique. ß-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Results of T cells of young (empty columns) and elderly (filled columns) donors are shown. Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Left panel shows the CD45/ß-actin expression (**, p < 0.01), and right panel shows CD45 activity, in optical density units, of T cell lysate immunoprecipitates determined using 4-nitrophenyl phosphate as a substrate. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. (C) The CD45RA/CD45RO ratio in T cells from young and elderly is shown (top panel). CD45RO activity (middle panel) and CD45RA activity (bottom panel) shown in optical density units in T cell lysate immunoprecipitates determined using 4-nitrophenyl phosphate as a substrate. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. The protein transferred to nitrocellulose membranes were stained with Ponceau to further verify that similar amounts of protein had been measured for each point of activity. Data are represented as the mean ± SD. Asterisks correspond to statistical significance (Student’s t-test) for p < 0.05 (*) and p < 0.01 (**). Data are representative of one of 20 independent experiments.

Fig 3: Characterization of bone marrow-derived mesenchymal stem cells: (a) the adherent cells appeared fusiform in shape with an elliptical nucleus (arrow); (b) BM-MSCs differentiated into adipocytes and stained with Oil Red O exhibited intense cytoplasmic staining, signifying the accumulation of lipid vacuoles (head arrow); (c) BM-MSCs differentiated into chondrocytes, exhibiting positive Alcian Blue staining (wavy arrow); (d) BM-MSCs differentiated into osteocytes and stained with Alizarin red staining, indicating clusters of calcium depositions (thick arrow); (e) FACS analysis of isolated stem cells showing that the bone marrow-derived mesenchymal stem cells were strongly positive for CD105, CD29, CD44, and CD90 markers but negative for CD34 and CD45 markers.

Fig 4: S100A4 exists in both the intima and media of the human carotid atheroma. A, A human carotid endarterectomyspecimen immunostained with anti‐S100A4 (S100 calcium‐binding protein A4) antibody. S100A4 was expressed in both the intima and media of the human carotid atheroma. S100A4‐positive cells were mainly expressed in the plaque shoulder and marginal areas. Scale bar: 1000 μm. B, Human carotid endarterectomy specimens stained with Oil Red O and anti‐S100A4 antibody. The left image was obtained using a phase‐contrast microscope; the center image was obtained using a fluorescence microscope (red: Oil Red O, green: S100A4, blue: nuclei); and the right image is a merged image of the phase‐contrast and fluorescence images. Images of the plaque shoulder and marginal areas are shown in the upper and lower sections, respectively. Foam cells accumulated around the necrotic core of the plaque shoulder, most of which were positive for S100A4. Similarly, in the plaque marginal area, accumulation of S100A4‐positive foam cells was observed between the expanded elastic laminae. Scale bars: 100 μm (low‐power field) and 50 μm (high‐power field). C, Human carotid endarterectomy specimens stained with anti‐S100A4 and anti‐SMemb antibodies (left: red, S100A4; green, SMemb; blue, nuclei); anti‐S100A4 and anti‐CD68 antibodies (center: red, S100A4; green, CD68; blue, nuclei); and anti‐S100A4 and anti‐CD45 antibodies (right: red, S100A4; green, CD45; blue, nuclei). S100A4 was found to colocalize with SMemb (left) and CD68 (center), but not CD45 (right). Scale bars: 50 μm. D, Samples stained with anti‐S100A4 and anti‐MMP9 antibodies (red, S100A4; green, MMP9). The upper image shows a plaque neointima, and the lower image, a plaque marginal area. In both these areas, S100A4‐positive cells were found to express MMP9. Scale bars: 50 μm. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; MMP, matrix metalloproteinase; and SMemb, embryonic form of smooth muscle myosin heavy chain.

Fig 5: Binding of IKKβ by wild-type and mutant B14 proteins. 293T cells were co-transfected with HA-tagged IKKβ and FLAG-tagged wild-type B14 or mutant Y35E, F130K, L126E, or T31K or the negative control VACV E3 protein. After 24 h, lysates from these cells were immunoprecipitated using either monoclonal anti-HA antibody or an isotype-matched control antibody against CD45 (iso) (A) or monoclonal anti-FLAG antibody (B and C). The immunoprecipitated (IP) and input proteins were resolved by SDS-PAGE on a 12% gel and then analyzed by immunoblotting (IB) with the indicated antibodies. Size markers are indicated on the left (in kilodaltons), and the identity of the protein bands is indicated on the right. The asterisk marks the position of the IgG light chain. C, after immunoprecipitation using anti-FLAG antibody, the amount of HA-IKKβ that co-precipitated with the wild-type or mutant B14 proteins was measured by quantitative immunoblotting (LI-COR; blot shown in supplemental Fig. S2). For each protein, the amount of co-precipitated IKKβ was normalized to the amount of IKKβ protein in the input lysates and then expressed relative to wild-type B14.