Figure 2. Expression of GFP constructs in primary human monocytes.
Elutriated primary human monocytes (5×106/group) were untransfected or transfected using DC solution either in absence of any plasmid DNA or in presence of pmaxGFP (Fig. 2A and Fig. 2C), GFP-PKCβ-WT (Fig. 2C) or GFP-PKCβ-DN (Fig. 2C) mutant plasmid (2µg) as described in Materials and methods. After nucleofection cells were kept in MNM either for different time intervals as indicated (Fig. 2A) or for 24 h (Fig. 2C) and then whole cell extracts were prepared. Equal amounts of lysate proteins were resolved by SDS-PAGE for immunoblotting with monoclonal GFP antibody to check the expression of both GFP and GFP-tagged PKCβ. The same blot was then stripped and further reprobed with anti-PKCβ to assess equal loading. After nucleofection with pmaxGFP vector (2µg), primary human monocytes were kept in MNM for 24 h and observed under brightfield and fluorescence (Fig. 2B).