Fig. 6.

Effect of β-Arrestin RNAi on UCN-II-Induced CRH-R2β Desensitization and Internalization in 293-R2β Cells

A, Cells were transfected with 1 nmol siRNA oligonucleotides for β-arrestin1 or -2 or scrambled oligonucleotide, and the effect on β-arrestin1 or -2 protein expression was determined in cell lysates by immunoblotting with a pan-arrestin antibody. Level of expression of GAPDH was also determined by immunoblotting (bottom panel). B, CRH-R2β homologous desensitization (induced by pretreatment with 100 nm UCN-II for 30min) was determined by measurement of UCN-II-induced cAMP production. Data represent the mean ± sem of two estimations from three independent experiments. *, P < 0.05 compared with cells without UCN-II pretreatment; +, P < 0.05 compared with control cells. C, Alternatively, internalization of UCN-II CRH-R2β was monitored by indirect confocal microscopy using specific primary antibodies and Alexa Fluor 594 secondary antibody for CRH-R2β (red). Identical results were obtained from four independent experiments. At least 20 individual cells in five random fields of view were examined. Inset, Fluorescence intensity of cytoplasmic CRH-R2β distribution, from 20 individual cells in five random fields of view was examined, and the sum of fluorescence intensity of cytoplasmic (distance 4–18 μm) fluorescence was measured by the ImageJ software. Results are expressed as the mean ± sem of three estimations from 20 individual cells. *, P < 0.05 compared with control (scrambled sequence) cells.