PLD2 activity is required for constitutive mGluR internalization. a, RT-PCR analysis of PLD2 mRNA expression in HEK 293 cells either before (control) or after the transfection of cells with 100 nm of either PLD2 siRNA or scrambled siRNA (scrambled) for 48 and 72 hr. RT-PCR of actin mRNA under identical conditions is provided as a control. The bar graph shows the mean ± SE for four experiments for PLD2 mRNA expression compared with β-actin mRNA expression after treatment with PLD2 and scrambled siRNA constructs ^*p < 0.05 versus untreated cells. b, Effect of cotransfecting 100 nm of either PLD2 or scrambled siRNA with 1 μg of FLAG-mGuR1a plasmid on the constitutive mGluR1a internalization at 48 and 72 hr after transfection. ^*p < 0.05 versus cells transfected with FLAG-mGluR1a alone (control). Data are presented as the mean ± SE of four independent experiments. c, Constitutive FLAG-mGluR1a internalization in HEK 293 cells after the incubation of the cells with and without 1% 1-butanol and 1% 2-butanol for 30 min at 37°C. ^*p < 0.05 versus untreated cells transfected with FLAG-mGluR1a alone (control). Data are presented as the mean ± SE of four independent experiments. In b and c, internalization was calculated as the percentage of loss of cell surface immunofluorescence after the incubation of primary FLAG antibody-labeled cells for 30 min at 37°C as measured by flow cytometry. d, Internalization of Alexa Fluor 555-conjugated FLAG antibody in cerebellar neurons transfected with FLAG-mGLuR5a and treated with either 1% 1-butanol or 1% 2-butanol. Data are presented as the mean ± SE of 10 independent experiments. ^*p < 0.05 versus cells maintained on ice. e, Internalization of Alexa Fluor 555-conjugated FLAG antibody in cerebellar neurons transfected with both FLAG-mGLuR5a and 100 nm of either PLD2 or scrambled siRNA. Data are presented as the mean ± SE of nine independent experiments. ^*p < 0.05 versus cells maintained on ice.