|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 4. Site-directed mutagenesis of the Runx2 retroviral vector. (A) Schematic diagram of the Runx2 retroviral expression vector and its mutated derivatives, including (a) Runx2-WT control, (b) Runx2-125Gly (mimicking constitutive dephosphorylation), and (c) Runx2-125Glu (mimicking constitutive phosphorylation). Primary dermal fibroblasts were transduced with Runx2-WT, Runx2-125Gly or Runx2-125Glu retrovirus and cultured in osteogenic media with (+) and without (–) 10 nM DEX. (B) Retroviral transduction efficiency was determined at 3 days post-transduction by flow cytometry detection of eGFP expression. Unmodified cells were used to detect autofluorescence. (C) Runx2 mRNA expression was assessed by quantitative RT-PCR at 3 days post-transduction and expressed on a logarithmic scale [mean + s.e.m., n=12; ANOVA: P<1E-11; **different from unmodified and DEX-only controls (P< 0.05)]. Detection limit determined by reactions without cDNA is shown as a dotted line. (D) Runx2 protein levels were examined at 7 days post-transduction by western blotting of whole-cell lysates with a polyclonal antibody against Runx2. GAPDH was used as a loading control. Blot is representative of data from two separate experiments in triplicate.
|
