FIGURE 4.

nNOS ubiquitination by the DE52-retained fraction of reticulocyte lysate requires Hsp70 and CHIP. A, methylene blue (MB) inhibits nNOS ubiquitination. Purified nNOS was incubated for 1 h at 37 °C with the DE52-retained fraction of reticulocyte lysate, ATP, GST-ubiquitin, and the indicated concentrations of methylene blue. In addition, 8 μm purified Hsp70 was added to a sample of the DE52-retained fraction containing 1 μm methylene blue. Samples were Western-blotted by probing with anti-nNOS. Lane 1, incubation time 0; lanes 2–7, incubation time 1 h. For the bar graph, the relative amount of monoubiquitinated nNOS (nNOS-Ub) in replicate experiments was determined by scanning and expressed as % of the 1-h control without methylene blue. The values are the mean ± S.E. (n = 3). Asterisks over the columns denote significantly different from control, and asterisks over the line denote that condition 7 is significantly different from condition 5. The top inset shows controls without GST-Ub and without added ATP (note: the stock, DE52-retained fraction contains 0.5 mm ATP.) The bottom inset (top row) shows an aliquot of input (INP) ubiquitinated nNOS of which 50 μl aliquots were immunoadsorbed with nonimmune (NI) or α-GST (I) IgG and immunoblotted (WB) with α-nNOS. The bottom row shows the effect of 3 μm methylene blue on samples immunoadsorbed with α-GST and immunoblotted with α-nNOS. B, CHIP is the major E3 ligase for nNOS ubiquitination. Purified nNOS was incubated with the DE52-retained fraction of reticulocyte lysate as above but in the presence of 1% nonimmune serum or 1% anti-CHIP serum. Lane 1, incubation time 0; lanes 2–4, incubation time 1 h.