The PCR products were purified with a NucleoFast 96 PCR Plate (Macherey-Nagel). Cycle sequencing was performed using a BigDye Terminator v3.1 Cycle Sequencing kit and ABI 3100 genetic analyzer (Applied Biosystems). For sequencing the intron 6 of the MFG-E8 chromosomal gene, a DNA fragment was amplified by PCR using the following primers: (GGGACCTCTCCCTTGAGCAC and CCAGTTCGCACTGTCATTAC), and subjected to the cycle sequencing. The normal RO4929097 purchase and mutant (IVS 6-937) alleles of intron 6 in the human MFG-E8 gene were amplified from the genomic DNA of the SLE patient. The 1791 bp PstI-ApaI fragment carrying intron 6 was used
to replace part (52 bp of PstI-ApaI DNA fragment) of the human MFG-E8
cDNA in pEF-BOS-hMFG-E8-Flag 15, and the product was verified by DNA sequencing. The minigene was introduced into HEp-2 cells by lipofection using Fugene 6 (Roche). Briefly, 1×105 cells were transfected with 0.5 μg DNA and cultured overnight in DMEM containing 10% FCS. After treating the cells selleck compound for 2 h with 100 μg/mL cycloheximide, the total RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen). The cDNA was synthesized with high capacity RNA-to-cDNA kit (Applied Biosystems) and subjected to PCR with the following primers: Cry-S, 5′-GCAGGACGATGATCTGCCTA-3′; Ex7-S, 5′-CGTAACTTTGGCTCTGTCCA-3′; and Flag-AS 5′-CGTCCTTGTAGTCGCTAGCA-3′. To prepare human rMFG-E8, the cDNA for the Flag-tagged human
MFG-E8 was inserted into pTRE2 expression vector (Clontech), and introduced into HAM3 cells, a HeLa tet-on cell line 36, with a vector carrying the hygromycin-resistance gene. After selection with 0.5 mg/mL hygromycin, the transformant clones that produced MFG-E8 in a PRKACG doxycycline-induced manner were selected. To produce MFG-E8, the transformants were treated with 1 μg/mL doxycycline in DMEM containing 1% FCS, and the secreted MFG-E8 was purified using anti-Flag M2 affinity gel (Sigma-Aldrich). To analyze the sugar moiety attached to human MFG-E8, rMFG-E8 (35 ng protein) was incubated at 37°C for 1 h with 0.1 unit of neuraminidase (Nacalai) or 500 units of PNGase F (New England Biolabs) and subjected to 10% SDS-PAGE, followed by Western blotting using an anti-Flag mAb. The binding of hMFG-E8 to phosphatidylserine was determined by the solid-phase ELISA as described 20. The Biacore technology using BiacoreX (GE Healthcare) with a HPA sensor chip was utilized to determine the dissociation constant for the binding of hMFG-E8 to phosphatidylserine according to Saenko et al. 37. Phagocytosis was assayed as described previously 7 with NIH3T3 cell transformants expressing αvβ3 integrin as phagocytes and apoptotic CAD−/− thymocytes as preys. After engulfment, the cells were stained with TUNEL using an ApopTag peroxidase in situ apoptosis detection kit (Chemicon).