Western blot analysis of sTNFRII-gAD in culture supernatants using monoclonal antibody against adiponectin (A) and sTNFRII (B).

<p>Lane 1, molecular weight markers (kDa); Lane 2, reducing conditions; Lane 3, nonreducing conditions.</p

<i>In vivo</i> migratory responses are impaired in mDia1-deficient mice.

<p>(<b>A</b>) Effector T cells were generated by <i>ex</i> <i>vivo</i> stimulation of Ova-specific T cells from mDia1^-/- OT-II or OT-II TCR mice (CD45.2⁺) with Ova peptide-loaded splenocytes for 6 days and the effector cells then injected into B6.SJL recipient mice (CD45.1) followed by intradermal challenge with OVA-peptide in incomplete Freund’s adjuvant. The accumulation of donor CD45.2 mDia1^-/- and wild-type effector T cells in Ova antigen-challenged skin was determined by immunostaining of single skin cell suspensions with PE-conjugated-anti-CD45.2 and Apc-conjugated-anti-CD4 antibodies. (B-G) Intranodal T cells migration. Naive CD4⁺ T cells purified from the spleens of mDia1^-/- and wild-type mice were labeled with CFSE or CMTMR and the mixture (1:1) of CFSE-labeled mDia1^-/- and CMTMR-labeled wild-type T cells intravenously injected into C57BL6 mice. Lymph nodes from recipient mice were imaged 24 hrs later by time-lapse two-photon confocal microscopy. The migratory parameters of migrating T cells in time were calculated from the movies using Velocity software. (<b>B</b>) Representative three-dimensional tracks of mDia1^-/- and wild-type T cells over a 20 min period. Each colored line represents a single T cell track. (<b>C</b>) Relative frequency of average velocity calculated from the net distance traveled during each time interval (displacement/time during a single time step) of a cell over 20 min; (<b>D</b>) Frequency of mean turning angles from which a cell deviates between successive time steps over 20 min; (<b>E</b>) Frequency of mean square displacement (left panel),a measure of the average distance a given particle in a system travels, and average mean square displacement over 20 min (right panel) are shown for wild-type and mDia1^-/- cells. (<b>F</b>) Mean meandering index, a measurement of directionality, was calculated by dividing the distance a cell traveled by the track length. (<b>G</b>) Motility coefficient (M) of mDia1^-/- and control T cells. M is a measure for how fast cells displace from their starting positions during a random walk process, analogous to the diffusion coefficient for Brownian motion (M=displacement²/6<i>t</i>). Data are expressed as the mean values ±SEM and are representative of at least 4 independent experiments. ∗∗ indicates <i>p</i><0.01, *** indicates <i>p<0.001</i>. </p

Biological activity of purified sTNFRII-gAD.

<p>An asterisksymbol (*) indicated a statistically significant difference (<i>p</i><0.05) between sTNFRII-Fc and sTNFRII-gAD. All assays were performed in triplicate, with error bars representing the standard error of the mean of the samples.</p

The metabolism of glucose and lactate of sTNFRII-gAD-expressing CHO-S cells in 3L and 5L fed-batch cultures in shaker bottles and AP30 bioreactor.

<p>(F: feed medium F001).</p

SDS-PAGE and western blot analyses of the purified sTNFRII-gAD.

<p>(A) SDS-PAGE analysis of sTNFRII-gAD purified by anion exchange chromatography. Lane 1:molecular weight markers (kDa); lanes 2, 3: The loaded protein samples were about 5.0 µg each loading in non-reducing and reducing conditions, respectively. (B) SDS-PAGE analysis of sTNFRII-gAD separated by HiLoad 16/60 Superdex 200 chromatography. Lane 1, protein molecular weight markers; Lane 2, multimeric sTNFRII-gAD (peak 1); Lane 3, trimeric sTNFRII-gAD (peak 2); Lane 4, monomeric sTNFRII-gAD (peak 3). The loaded protein samples were about 2.0, 5.0, and 3.0 µg, respectively. (C) Western blot analysis of purified trimeric sTNFRII-gAD under non-reducing/reducing conditions. Lane 1, molecular weight markers; Lanes 2 and 3 The loaded protein samples were about 5.0 µg and 3.0 µg in non-reducing and reducing conditions, respectively.</p

Comparison of the Growth and sTNFRII-gAD Fusion Protein Production of the Engineered Cells in Suspension Culture.

a<p>Estimated by BCA protein assay after anion exchange chromatography.</p>b<p>Determined by densitometry of gel (data not shown).</p><p>Comparison of the Growth and sTNFRII-gAD Fusion Protein Production of the Engineered Cells in Suspension Culture.</p

mDia1 is required for induction of MT plus-ends dynamics downstream of LFA-1-ICAM-1 interaction.

<p>WT or mDia1^-/- T lymphoblasts were loaded onto ICAM-1(3µg/ml)-coated plates for 30 min, fixed and immunostained using FITC-conjugated anti-α-tubulin and Cy3-conjugated anti-EB1 antibody. (<b>A</b>) <i>Left</i>: Representative images of WT and mDia1^-/- T cells migrating on ICAM-1 show EB1 clustering with MTs plus ends in polarized T cells. The images shown in the far right panel are enlarged versions of the boxed areas shown in the middle panels. <i>Middle</i> <i>graph</i>: Quantitation of the fluorescence intensities of EB1 staining across the cell axis from uropod to the leading edge of polarized T cells (depicted by dashed arrow in far left image).<i>Right</i> <i>bar</i> <i>graph</i>: Quantitation of the number of cells among 100 cells assessed showing EB1 binding to the tips of growing MT plus-ends at the leading edge cell periphery. (<b>B</b>) <i>Left</i>: Representative images showing the distribution of APC in wild-type and mDia1^-/- T cells migrating on ICAM-1. <i>Right</i>: Quantitation of the number of cells among 100 cells counted showing APC clustering at the leading edge. (<b>C</b>) Lysates prepared from mDia1^-/-and WT T cells were subjected to SDS-PAGE and immunoblotted with anti-APC or anti-EB1 antibodies followed by anti-α-tubulin antibody. (<b>D</b>) Wild-type or mDia1^-/- T lymphoblasts were treated for the indicated times with 10μM cyclohexamide in the absence or presence of the proteosome inhibitor MG-132 (10μM). The cells were then fixed and permeabilized for immunostaining with anti-APC antibody followed by flow cytometric analysis. Data are expressed as the mean fluorescence intensity (MFI). Values represent mean ± SEM and all data for A-D are representative of 4 independent experiments. ∗∗∗, <i>p</i><0.001 by two-way ANOVA, NS: not significant. </p

Dot blot analysis of sTNFRII-gAD protein in 96-well plates at 24 h with monoclonal antibody against TNFRII.

<p>Lane 1∶100, 75, 50, 25, 12.5 and 6.25 µg/ml of sTNFRII-Fc; Lane 2, 3, 4∶1/1, 1/3 and 1/6 dilutions of the supernatants from the 1st, 2nd and 3rd hyper-expression clones.</p

Schematic diagram of the recombinant plasmid pMH3-sTNFRII-gAD.

<p>GC-rich element: GC-rich non-coding DNA fragments; sTNFRII-gAD: soluble TNF receptor II and globular domain of adiponectin; actin promoter: chick beta-actin promoter; polyA: rabbit globulin polyA signal.</p

Ultracentrifuge analysis of the trimerized sTNFRII-gAD.

<p>It gave a molecular weight of 165 kDa.</p