, ?g/ml; Sigma-Aldrich) followed by 6 days with IL-2 (20 ng/ml; PeproTech) for blasting. The A0.01 T cell (from H.T. He, Centre d'Immunologie de Luminy, Marseille, France) and Jurkat cell (ATCC) lines were maintained in complete RPMI medium. The CHO K1 cell line (ATCC) was cultured in complete HAM F-12 medium. Dermal fibroblasts, isolated and expanded from healthy and WHIM WT skin biopsies , were maintained in complete DMEM. EBV-transformed B cell lines derived from healthy individuals and P4 were cultured in complete RPMI medium supplemented with 50 ?M 2-mercaptoethanol. The marked B cell lymphopenia affecting P3 did not permit us to generate a B cell line. This study was approved by the Direction Médicale et Santé Publique, Institut Pasteur, and all subjects gave informed consent for this investigation. Transfection and functional assays, The human SMARTpool SCR (siCON- TROL non-targeting#1), GRK2, and GRK3 siRNA duplexes
, QIAGEN Sciences) and reverse transcribed (Superscript II, BD Biosciences Clontech) by extension of oligo(dT) priming using a template-switch primer 5?-AAGCAGTGGTATCAACGCAGAGTAC[T]20VN-3? (47) Amplification of oligo(dT)-primed cDNAs was performed by PCR (Advantage II pol40 cycles: 95°C 30 s, 68°C 3 min) using forward 5?-GGTTCCAAGTCCAATATGGCAA-3? and reverse 5?-ACTAG- GCATGTTCAGTGAAGGAGC-3? primers for LDH, Total cellular RNA was extracted using the RNeasy kit 5?-CAAGAT- GGCGGACCTGGA-3? and reverse 5?-GGGTTGGTGCAGCAGGA-3? primers for GRK2, forward 5?-CCGCCAAAGCTCGCCAAC-3? and reverse 5?-ATCAGATGCCTCATCCTCGTTCAC-3? primers for GRK3, forward 5?-GCTCCGTTGCTGACCGC-3? and reverse
, Sigma-Aldrich) for 0?4 h before RNA isolation. mRNA half-lives were determined by fitting exponential decay curves to experimental data points Primer pairs for quantitative RT-PCR were retrieved on the PrimerBank Web site PCR reactions were performed with SYBR Green (Applied Biosystems) detection using forward 5?-CTCGGGCCACTTCACTGAC-3? and reverse 5?-CAGTCCG- TACCTACCGATGTAT-3? primers for ??l-iduronidase (IDUA), forward 5?-ACTTCAGCGTGCATCGCAT-3? and reverse 5?-GCTTTTTGTCCAG- GCACTTCAT-3? primers for GRK2, forward 5?-AGCTGTAGAACACGTA- CAAAGTC-3? and reverse 5?-ATGTCACCTCGAAGGCTTTCA-3? primers for GRK3, and forward 5?-TAGCGAACACGGTGCTACTC-3? and reverse 5?-GCTGATGTGAGGGAACTGGA-3? primers for GRK6. We used the ABI 7300 Sequence Detection System (Applied Biosystems) with the following amplification scheme: 50°C 2 min, 95°C 10 min and 40 cycles: 95°C 15 s, 60°C 30 s, 68°C 40 s. The dissociation curve method was applied according to the manufacturer's protocol (60°C to 95°C) to ensure the presence of a single specific PCR product. Analysis was performed with the standard curve method, and results were expressed as GRK/IDUA ratios. IB analysis. Equivalent amounts of proteins (30 ?g/lane unless specified) were separated by SDS-PAGE on 4%?12% Tris-glycine polyacrylamide gels (Invitrogen), transferred to PVDF membrane and probed with the following monoclonal or polyclonal Abs: sheep anti-human LDH (Biodesign), rabbit anti-human ?-arrestin1 (clone E246; Epitomics), rabbit anti-serum to ?-arrestin2 (a gift from R, RNA synthesis and stability assays, cells were treated with 10 ?g/ml ActD rabbit anti-human GRK2 or -6, goat anti-human GRK3 (all from Santa Cruz Biotechnology Inc.), mouse anti-rat GRK2 Oppermann) Abs. Peroxidase-conjugated anti-mouse, antirabbit (both from Amersham Biosciences) and anti-goat (Vector Laboratories ) Abs were used to detect bound primary Abs by enhanced chemiluminescence . IB quantitation was done using an LAS-1000 CCD camera with Image Gauge 3.4 software
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