Cell culture and transfection
293T cells were grown in Dulbecco's modified Eagle's Medium (DMEM, Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS, EQUITECH-BIO, Ingram, TX). For transfection, 293T cells in 6-well tissue-culture plates at approximately 70% confluence were transfected with a total of 1 μg of plasmid vector DNA using the Fugene 6 transfection reagent (Roche, Mannheim, Germany) according to the manufacturer's instructions.
Constructs described below were used for over-expression of full-length Zizimin2 or its CZH2 domain. DNA coding full-length Zizimin2 or its CZH2 domain was cloned into pCNX2-HA using NotI site for full-length or EcoRI and XhoI for its CZH2 domain, which are referred to as HA-Zizimin2 expression vector, pCNX2-HA-Zizimin2, and HA-CZH2 domain expression vector, pCNX2-HA-CZH2.
Tissues or cells were lysed by the addition of RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitors (Roche, Mannheim, Germany) and homogenized on ice with a homogenizer. Insoluble materials were pelleted by centrifugation at 15,000 g for 20 min at 4°C, and protein in the supernatant was quantified by bicinchoninic acid protein assay (Pierce, Rockford, IL). Protein dissolved in SDS sample loading buffer (62.5 mM Tris-HCl (pH6.8), 5% 2-mercaptoethanol, 2% SDS, 5% sucrose, 0.002% bromophenol blue) was boiled for 5 min, subjected to SDS-PAGE using 5-20% or 8% polyacrylamide gel, and transferred to polyvinylidene fluoride membrane. Membrane was blocked in PBS containing 0.1% Tween-20 (PBST) and 1% non-fat milk for Zizimin1 and Zizimin2 or 5% non-fat milk for α-tubulin overnight at 4°C. Then, the membrane was probed for 2 h with Zizimin2 antibody (1:100) (214I9, ), Zizimin1 antibody (1:1000) (NB500-265, Novus Biologicals, Littleton, CO), and anti-α-tubulin antibody (1:2000) (Sigma-Aldrich, St. Louis, MO) in PBST containing 1% non-fat milk, washed three times with PBST, and incubated with peroxidase-conjugated secondary antibody (1:2000) (Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature (RT). After several washes with PBST, reaction was visualized with Immobilon Western (MILLPORE, Billerica, MA) for detection of Zizimin2, Zizimin1, and Cdc42 or ECL (ECL plus Western blotting Detection System, GE Healthcare, Piscataway, NJ) for α-tubulin.
Quantitative real-time RT-PCR
Quantitative real-time RT-PCR was carried out as described previously . The following gene-specific primers were used: mouse Zizimin2, 5'-TTG CCT TTT ATG GCC AGT CT-3' (sense) and 5'-GAG CGA ATT TTG GAT CAA GC-3' (antisense), mouse GAPDH, 5'-AAT GGT GAA GGT CGG TGT G-3' (sense) and 5'-GAA GAT GGT GAT GGG CTT CC-3' (antisense).
Cdc42 activation assay
For Cdc42 activation assay, we used the Cdc42 activation kit (Cytoskeleton, Denver, CO) according to the manufacturer's instructions.
293T cells and BMDC cultured on poly-lysine-coated coverslips were washed three times in PBS, fixed for 15 min in 4% paraformaldehyde at 37°C, washed in PBS, and permeabilized with 0.1% TritonX-100 in PBS for 5 min at RT. The coverslips were washed three times in PBS, blocked for 1 h with 0.2% bovine serum albumin in PBS, washed three times in PBS, and incubated for 1 h with 214I9 hybridoma culture supernatant (1:1) or 214I9 (1:100)  for Zizimin2 staining, Alexa488-conjugated phalloidin (1:500) (Invitrogen, Carlsbad, CA) for actin staining, or sheep anti-cdc42 (1:100) (Cytoskeleton, Denver, CO) for Cdc42 staining. Following three washes in PBS, coverslips were incubated for 1 h with Cy3-labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA), washed three times in PBS, and mounted onto microscope slides using Vectashield containing 4',6-diamino-2-phenylindole (DAPI, Vector Labs, Burlingame, CA) for nucleus detection. Images were acquired using a Fluoview laser scanning microscope (OLYMPUS, Tokyo, Japan).
Isolation of BMDC, pDC, and mDC
Bone marrow cells obtained from C57/BL6 mouse were maintained in RPMI1640 supplemented with 10% X63-GM cell culture supernatants as a source of granulocyte macrophage colony-stimulating factor (GM-CSF) or 50 ng/ml FMS-like tyrosine kinase 3 (Flt3) (R&D SYSTEMS, Minneapolis, MN) for 6 days to induce BMDC. On the 6th day, the cells were stimulated with 1 μg/ml lipopolysaccharide (LPS) and cultured for 2 more days. On the 8th day, the cells were stained with FITC-conjugated anti-CD11c antibody (BD Bioscience, Franklin Lakes, NJ) and CD11c-positive cells were sorted using a cell sorter, JSAN (Bay Bioscience, Kobe, Japan), which were defined as BMDC in this study. pDC (CD11c+, B220+, mPDCA-1+, Ly-6C+) and mDC (CD11c+, B220-) were sorted from BMDC using a cell sorter, JSAN (Bay Bioscience, Kobe, Japan). All animal procedures were approved by institutional reviews board at National Center for Geriatrics and Gerontology followed the guideline issued by Japanese Ministry of Health Labour and Welfare.
Isolation of B cells and T cells
Splenocyte suspension was prepared from mouse spleen and splenic B cells or T cells were isolated with MACS beads (Miltenyi Biotec, Gladbach, Germany), according to the manufacturer's instructions.