Figure 2. Role of specific siRNAs for β-arrestin1/2 in CCR5 regulation.

(a) Schematic representation of kinetics experiment and immunoblot analysis of β-arrestin1/2 and GAPDH (loading control throughout) in whole R5-SupT1-L23 cell-lysates nucleofected with siRNAs directed to β-arrestin1 (β1), β-arrestin2 (β2) or both and with NS-siRNA (negative control) for all time points. (b–d) Immunoblot analysis of phosphorylated and total CCR5 and ERK1 in whole-cell lysates harvested after treatment with anti CCR5 Ab Pos and appropriate controls (RANTES and CCR5 Ab Neg), at t0 (corresponding to 30′ of CCR5 modulating factors incubation, washing and additional 120′ of cells incubation in medium without stimuli (150′)) (b), t1 (corresponding to 48 h of factors incubation) (c), t2 (corresponding to 48 h of factors incubation, washing and a further 24 h of cells incubation in medium without stimuli) (d). Protein levels have been determined with densitometric analysis of the blot with the T.I.N.A. program and expressed as fold change over the appropriate housekeeping genes. Bar graphs represented mean ± SD of three independent experiments. Student’s t-test was performed and p values are shown. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (b,c right panels).