High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells (original) (raw)
Figure 2
Expression of Hsp70 (GC3 = 92%) and Hsc70 (GC3 = 46%)
(A) Three independent clones of pcDNA3-Hsp70-HA (GC3 = 92%) and six clones of pcDNA3-Hsc70-HA (GC3 = 46%) were used to transfect HeLa cells. 24 h following transfection the cells were harvested and the Hsc70-HA or Hsp70-HA protein levels were analyzed by Western blotting using an anti-HA antibody. An anti-GAPDH antibody was used as loading control. (B) Equal amounts of Hsp70 and Hsc70 mRNA were used as templates for in vitro translation in rabbit reticulocyte lysates in the presence of 35S-Methionine. The reaction was initiated by the addition of reticulocyte lysate to the translation mix and samples were removed in 2-min intervals into SDS sample buffer. The reaction products were analyzed by SDS-PAGE and autoradiography. (C–E) HeLa cells were transfected with equal amounts of pcDNA3-Hsp70-HA or pcDNA3-Hsc70-HA plasmids. After 24 h, total cellular RNA was isolated and analyzed by qRT-PCR. The graphs represent Hsp or Hsc70 (C), neo (D), and GAPDH (E) mRNA amounts. Hsp70, cells transfected with pcDNA3-Hsp70-HA; Hsc70, cells transfected with pcDNA3-Hsc70-HA; control, untransfected cells. The mRNA amounts were normalized to the amounts in the Hsp70-transfected cells. The error bars represent standard deviations from three to four independent transfections.