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. 2004 Oct 6;24(40):8752–8761. doi: 10.1523/JNEUROSCI.3155-04.2004

Figure 8.

Figure 8.

Effect of ARF1, ARF6, and PLD1 on the constitutive mGluR1a internalization. a, The effect of overexpressing wild-type, constitutively active (Q67L), and dominant-negative (T31N) ARF1 protein on constitutive FLAG-mGluR1a internalization in HEK 293 cells. b, Representative immunoblot showing the lack of coimmunoprecipitation of ARF1 with FLAG-mGluR1a from HEK 293 cells. c, The effect of overexpressing wild-type, constitutively active (Q67L), and dominant-negative (T27N) ARF6 protein on constitutive FLAG-mGluR1a internalization in HEK 293 cells. d, Representative immunoblot showing the lack of coimmunoprecipitation of ARF6 with FLAG-mGluR1a from HEK 293 cells. e, The effect of overexpressing wild-type PLD1 and a catalytically inactive (K898R) PLD1 mutant on constitutive FLAG-mGluR1a internalization in HEK 293 cells. f, Representative immunoblot showing the lack of coimmunoprecipitation of PLD1 with FLAG-mGluR1a from HEK 293 cells. HEK 293 cells were transiently transfected with 7 μg of FLAG-mGluR1a and 7 μg of empty vector or myc epitope-tagged ARF1, ARF6, and PLD1 plasmid cDNAs. The internalization data represent the mean ± SE of five independent experiments, and the coimmunoprecipitiation experiments were repeated on three independent occasions from 500 μg of HEK 293 cell lysate. g, Representative confocal micrographs demonstrating the lack of colocalization between FLAG-mGluR1a (red) and PLD1-YFP (green) after the constitutive internalization of Alexa Fluor 555-conjugated antibody-labeled FLAG-mGluR1a for 60 min at 37°C. The data are representative of four independent experiments. HEK 293 cells were cotransfected with 5 μg each of Flag-mGluR1a and PLD1-YFP. Lysates correspond to 100 μg of total HEK cell lysate. Scale bar, 10 μm.