Journal: Journal of Bone and Mineral Research
Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B
Figure Lengend Snippet: Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .
Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).
Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Construct, Amplification, Polymerase Chain Reaction, FLAG-tag, Synthesized, Agarose Gel Electrophoresis, Transfection, Stable Transfection