RT-PCR experiment to analyze IL-6, IL-8, and IL-15 expression in
wnt5a-transfected (transient) normal synovial
fibroblasts and determination of transfection efficiency.
(A) Lane 1, DNA standard PhiX174; lanes 2–5, IL-6-,
IL-8-, IL-15-, and G3PDH-specific RT-PCR products from
wnt5a-pcDNA3-transfected normal synovial fibroblasts;
lanes 6–9, IL-6-, IL-8-, IL-15-, and G3PDH- specific RT-PCR products
from empty vector transfected normal synovial fibroblasts; lanes
10–13, IL-6-, IL-8-, IL-15-, and G3PDH-specific RT-PCR products from
untransfected normal synovial fibroblasts. (B) Bar graph
showing fold increase in IL-6, IL-8, and IL-15 gene expression
(measured by PCR product intensity) on transfection of normal synovial
fibroblasts with wnt5a expression construct. Fold
increase with wnt5a is compared with the effects
produced by transfection by the empty vector. This represents an
average of five different experiments. For each experiment, the same
amounts of RNA were used from the wnt5a- and empty
vector-transfected cells. G3PDH expression was used an internal
control. (C) Western blot of lysate from
wnt5a-HA-transfected synovial fibroblasts. Lane 1, ≈60
kDa wnt5a-HA protein from
wnt5a-HA-transfected synovial fibroblasts; lanes 2 and
3, no band observed from untransfected synovial fibroblasts.