Association of NGF receptors and cytoskeletal elements with DRMs under different experimental conditions.

<p>A) Western blots showing TrkA and p75^NTR in floating DRMs prepared after 10 min NGF treatment and using nuclease (Benzonase) rather than sonication to break up nucleic acids prior to equilibrium density gradients. Western blots of flotation equilibrium gradients of detergent-resistant fraction were probed with anti-TrkA, -pTrkA, -SHP-1, -p75^NTR, and -tubulin (indicated). Blots include the detergent-sensitive (P1M) fraction and size standards (S) to the left of DRM gradient fractions. B) Left: ¹²⁵I-NGF in DRM without (open squares) and with (closed circles) in vitro reactions with ATP. Right: Quantification of chemiluminescent signals from western blots is compared to ¹²⁵I-NGF for Benzonase-treated samples as in A. Fraction number is plotted on the x-axis of plot on the left; fraction 1 has the highest density. Signals from western blots were quantified and plotted vs. density together with ¹²⁵I-NGF (closed circles) on the right. The y-axis for TrkA (closed squares), p75^NTR (open circles), SHP-1 (open squares), and tubulin (closed triangles) is arbitrary units (chemiluminescent pixel volume). C) Western blots showing actin in floating DRMs prepared as in A, with and without in vitro reactions with ATP (–, +ATP). Data obtained under these conditions for TrkA, tubulin, and actin were quantified and plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g002" target="_blank">Figure 2</a>. After 10 min internalization, in vitro reactions with ATP caused a significant increase of TrkA (p<0.01) and NGF and tubulin (p<0.001) in floating DRMs. A decrease in actin (+ATP) was noted but was not statistically significant.