Figure 1

NGF promotes the interaction of Gβγ with MTs and stimulates MT assembly. PC12 cells were treated with 100 ng/mL of NGF for three consecutive days. Microtubules (MTs) and soluble tubulin (ST) fractions (A–D), or cell lysates (E) were prepared as described in the methods. (A–C) Equal amounts of proteins from MT or ST fractions were subjected to co-immunoprecipitation (tubulin and Gβγ) using anti-tubulin (A) or anti-Gβ (B) followed by immunoblot analysis (Gβ and tub) of immunoprecipitates (IP) and supernatants (SUP) as indicated in the figures. Control experiments include immunoprecipitation in the absence of a primary antibody (No Ab) or in the presence of non-specific rabbit or mouse IgG (IgG). Immunoprecipitation of tubulin or Gβ resulted in co-immunoprecipitation (CO-IP) of tubulin and Gβ. Protein bands (IP) were quantitated and expressed as NGF-induced increase in CO-IP (C). Bar graph shows the mean ± standard error from 3–5 (N) independent experiments as indicated (C). (D) Polymerized (MT) and free tubulin (ST) contents as well as the association of Gβ in MT/ST fractions were analyzed by immunoblotting (IB) (left panel). Bar graph represents MT assembly (percent of tubulin in MT) or the percent Gβγ in MT fractions (D, right panel) from five independent experiments (mean ± standard error). Loading control include re-probing the blots with anti-actin. (E) Representative immunoblots show that NGF does not alter tub or Gβ immunoreactivity in cell lysates (left panel). Loading control include actin. The NGF effect on the increase in co-immunoprecipition of tub and Gβγ (using anti-tub antibody) is shown in the right panel. *p < 0.05; ***p < 0.001.