Journal: The Journal of Neuroscience

Article Title: Phosphatidylinositol 4,5-Bisphosphate Modifies Tubulin Participation in Phospholipase Cβ1Signaling

doi: 10.1523/jneurosci.22-05-01668.2002

Figure Lengend Snippet: Figure 4. PIP2 does not increase Gq transactivation by tubulin. A, The effects of carbachol and PIP2 on the membrane association of tubulin are additive. Recruitment of tubulin to the membrane was studied using increasing concentrations of FM-tubulin. Membranes from Sf9 cells ex- pressing m1 muscarinic receptors, Gq, and PLC1 were incubated with carbachol and FM-tubulin (at the indicated concentrations), with or without PIP2. SDS-PAGE (50 g of membrane protein in each lane) was followed by measurement of the fluorescence of membrane-associated tubulin with a fluorescence imaging system (Storm 840; Molecular Dy- namics). The results represent one of three similar experiments per- formed in triplicate. Open circles, Membranes treated with 1 mM carba- chol; filled circles, membranes treated with 1 mM carbachol and 30 M PIP2. B, PIP2-assisted recruitment of tubulin–[ 32P]AAGTP to the mem- brane is concentration-dependent. Membranes from Sf9 cells containing m1 muscarinic receptors, Gq, and PLC1 were incubated with 1 mM carbachol, 1 M tubulin–[ 32P]AAGTP, and increasing concentrations of PIP2, as described in Figure 1. After SDS-PAGE (50 g of membrane protein in each lane) 32P-labeled protein bands were measured by phos- phorimage analysis. One of three identical experiments done in triplicate with similar results is shown. C, Carbachol-evoked Gq transactivation by tubulin is not affected by PIP2. The experiments were done as described in A, except that tubulin–[ 32P]AAGTP was used. Proteins were resolved by SDS-PAGE (50 g of membrane protein in each lane), and the radioactivity of the Gq bands ([ 32P]AAGTP was transferred from tubu- lin) was measured by phosphorimage analysis (Storm 840; Molecular Dynamics). One of five independent experiments done in triplicate with similar results is shown. Open circles, Membranes treated with 1 mM carbachol; filled circles, membranes treated with 1 mM carbachol and 30 M PIP2. D, The increased membrane association caused by PIP2 was not linked to Gq transactivation. The percent ratios of [ 32P]AAGTP-labeled Gq and tubulin–[ 32P]AAGTP at the various PIP2 concentrations are derived from the experiment described in B. One of three identical experiments done in triplicate with similar results is shown. Control values for B and D represent the amount of tubulin associated with the plasma membrane in the absence of carbachol or PIP2.

Article Snippet: The spots containing GTP or GDP standards were visualized with a UV lamp, and the plate was exposed to film for autoradiography or subjected directly to phosphorimage analysis (Storm 840; Molecular Dynamics).

Techniques: Membrane, Incubation, SDS Page, Imaging, Concentration Assay, Labeling, Radioactivity, Derivative Assay, Control, Clinical Proteomics