Journal: PLoS ONE

Article Title: Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth

doi: 10.1371/journal.pone.0018758

Figure Lengend Snippet: A. Coomassie stain of purified vaults fractionated on a 4–16% SDS-PAGE. Lane 1, molecular weight markers. Lane 2, mCCL21-mCherry-INT/CP-MVP vaults. The 97-kDa MVP band is indicated by the arrow. Lane 3, hCCL21-INT/CP-MVP vaults. Lane 4, CP-MVP vaults. B. Western blot analyses of purified vaults to confirm the presences of MVP or the indicated INT fusion proteins (arrows). Lane 1, mCCL21-mCherry-INT/CP-MVP vaults were immunoblotted with anti-MVP antibody. Lane 2, mCCL21-mCherry-INT/CP-MVP vaults were immunoblotted with anti-INT antibody. Lane 3, hCCL21-INT/CP-MVP vaults were immunoblotted with anti-INT antibody. C. Negative-stained electron micrographs of purified vaults. CP-MVP vaults (left), mCCL21-mCherry-INT/CP-MVP vaults (center), hCCL21-INT/CP-MVP vaults (right).

Article Snippet: Primary antibodies for western blot analyses were rabbit anti-MVP antibody (1/1000 dilution) or rabbit anti-VPARP antibody (1/500 dilution) and secondary goat anti-rabbit HRP-antibody (1∶2000 dilution) (Amersham).

Techniques: Staining, Purification, SDS Page, Molecular Weight, Western Blot

Journal: PLoS ONE

Article Title: Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth

doi: 10.1371/journal.pone.0018758

Figure Lengend Snippet: A. CCL21-vaults increased the migration of T2 cells. 2.0×10 5 T2 cells were plated in serum-free medium in the upper chamber. mCCL21-mCherry-INT/CP-MVP vaults (200 ng/ml or 600 ng/ml), recombinant CCL21 (600 ng/ml), control vaults (CP-MVP vaults 600 ng/ml) or neutralizing anti-CCL21 recombinant antibody (5 µg/ml) were added to the lower chamber of the wells. Following 2 hr incubation, migration of T2 cells were analyzed by flow cytometry. mCCL21-vaults effectively increased the T2 migration as compared with control. Anti-CCL21 neutralizing Ab to CCL21 abrogated the increase in T2 migration suggesting that CCL21 in the vault predominantly mediate the chemotatic migration of T2 cells. Data in the panel are representative of 2 independent experiments. Bars; Mean ± SEM, *p<0.05 between the mCCL21-vaults and control vaults or anti-CCL21 antibody treatment groups. B. mCCL21-vaults enhanced DC APC activity and blocking CCL21 reversed the increase in APC activity. B3Z cells (1×10 5 cells/200 µl/well) were co-cultured with DC 2.4 (5×10 4 cells/200 µl/well) and ovalbumin (350 µg/ml) in the presence or absence of mCCL21-vaults (200 ng/ml) and anti-CCL21 antibody (5 µg/ml) or control antibody (5 µg/ml goat IgG) for 24 hrs. Control vaults were used at a concentration of 200 ng/ml. T cell activation status was measured by IL-2 production by ELISA. Data are representative of 2 independent experiments. Bars; Mean ± SEM, *p<0.05 between the mCCL21-vaults and control vaults or anti-CCL21 antibody treatment groups.

Article Snippet: Primary antibodies for western blot analyses were rabbit anti-MVP antibody (1/1000 dilution) or rabbit anti-VPARP antibody (1/500 dilution) and secondary goat anti-rabbit HRP-antibody (1∶2000 dilution) (Amersham).

Techniques: Migration, Recombinant, Control, Incubation, Flow Cytometry, Activity Assay, Blocking Assay, Cell Culture, Concentration Assay, Activation Assay, Enzyme-linked Immunosorbent Assay