Effect of Gβγ-binding peptides, GRK2i, and mSIRK on MTs and Gβγ organization and neurite outgrowth. (A) PC12 cells were treated with 100 ng/mL of NGF for two consecutive days. Subsequently, 5 μM GRK2i was added to the media and the cells were incubated for 10, 30, and 60 min as indicated. The cells were then fixed and double labeled with anti-tubulin (red) and anti-Gβ (green) antibodies and processed for confocal microscopy. DAPI was used for nuclear staining (blue). Control cells exhibit typical neuronal morphology, displaying long neurites. Gβγ is shown to co-localize with tubulin/MTs along the neuronal processes (solid yellow arrow) but not at the tip of the neurites (green arrowheads), where Gβγ immunostaining is predominant. Inhibition of Gβγ signaling by incubation with GRK2i causes neurite damage, microtubule disruption, and alters the Gβγ-tubulin co-localization pattern as shown in the enlarged images in the white boxes (f’–h’, and f”–h”). To test the effect of mSIRK, PC12 cells were treated with mSIRK (1 μM) for 2 h, followed by 1-day treatment with NGF. Scale bars are 20 μm. (B–C) PC12 cells were treated with GRK2i or mSIRK as described above, followed by fixing and processing for confocal microscopy. Using Zeiss ZEN software, neurites were traced and measured, and average neurite length and percent of cells bearing neurites were determined. Differences between experimental conditions were assessed by one-way ANOVA. *p < 0.05; **p < 0.01.