Fig. 1.

A) Differential interference contrast image of a hippocampal culture from a postnatal day 4 rat pup treated with 10 μM control morpholino oligos and 2 μM Endo-Porter peptide, and maintained in culture for 96 hours. B) Fluorescent image of A; oligos were tagged with carboxyfluorescein for visualization. C) Western blot of proteins isolated from either a control culture (lane 1), a culture treated for 24 hours (lane 2) or 48 hours (lane 3) with 2 μM Gα_q oligos. Protein bands were visualized with Gα_q antibodies and β-tubulin antibodies as a loading control. Gα_q band densities were calculated by dividing the integrated optical density of the Gα_q band by the integrated optical density of the β tubulin band to control for loading and expressed as percent of control. D) Western blot analysis of 6 different preparations from cultured neurons treated for 48 hours with nonsense oligos (N=3, NS MO) or Gα_q oligos (N=3, Gq MO). The data were analyzed as in C, with the average of the nonsense oligo treated preparations defined as 100%. Data are expressed as mean±SEM. The inset is an example of Gα_q expression in control tissue compared to cells treated with nonsense morpholinos to demonstrate that the nonsense morpholinos do not cause nonspecific knockdown of Gα_q. E) Western blot analysis of Gα_q and Gα₁₁ G-protein in the neonatal hippocampus. Analysis was performed using tissue from the superior region of the hippocampus and antibodies against either Gα_q or Gα₁₁ proteins. The left panel shows molecular weight markers (same experiment but exposed to film for 1 sec). To visualize bands labeled with Gα₁₁ antibody, 10 times more protein was loaded (right panel) than for lanes labeled with Gα_q antibodies (center panel) and the exposure time was increased to 10 minutes, compared with 3 minute exposure for bands labeled with Gα_q antibodies.