Gβγ co-localizes with MTs in the neuronal processes in NGF-differentiated PC12 cells. PC12 cells were treated with and without NGF (control). (A) The cells were then fixed and double labeled with anti-tubulin (red) and anti-Gβ (green) antibodies as indicated in the methods. Areas of overlay appear yellow. The enlarged image of the white box (c) shows co-localization of Gβγ with MTs in the perinuclear region (c’). The white box on the lower panel (f’) shows the enlarged growth cone, with Gβγ co-localizing with tubulin along the neuronal process and in the central portion of the growth cone, while the neuronal tips show predominant Gβγ immunostaining. The solid yellow arrow indicates neuronal processes, and the broken yellow arrow indicates cell body. Green arrowhead indicates only Gβ labeling (not tubulin) at the neuronal tips. The scale bars in “a–c” and “d–f” are 20 μm and 50 μm, respectively. (B) Co-localization of Gβγ with MTs in the neuronal processes was quantitatively assessed using Zeiss ZEN software. A representative image of a region of interest (neuronal process) of an NGF-differentiated PC12 cell is shown. (C) A representative scattergram depicting co-localization of Gβγ with MTs along the neuronal process is shown. (D) Representative Western blots (using PC12 whole-cell lysates) showing the specificity of the anti-Gβ (left) and anti-tubulin (right) antibodies that were used for immunofluorescence.