Mouse embryonic fibroblasts (MEFs) with or without β-arrestin

2 were generous gifts from Dr Robert Lefkowitz, Duke University Medical Center. The human embryonic kidney 293 (HEK293) cells stably transfected with hTLR4 (HEK293/TLR4) or hTLR2 (HEK293/TLR2) were kindly provided by Dr Evelyn A. Kurt-Jones of the University of Massachusetts Medical School. β-Arrestin 2 full-length vector, short hairpin RNA (shRNA) vector and IWR-1 solubility dmso corresponding cloning vectors were generous gifts from Dr Gang Pei, Shanghai Institutes for Biological Sciences, China. The plasmids pcDNA3-GSK3β (S9A) and pcDNA3-GSK3β (K85A) were kindly provided by Dr Michael Martin (University of Louisville School of Dentistry, Louisville, KY). HEK293 and HEK293/TLR4 cells (3 × 105/dish) were seeded on 35-mm dishes 24 hr before transfection. Transfection was performed with 1 μg vector using LipofectAMINE 2000 reagent (Invitrogen Corporation, Carlsland, CA) according to the manufacturer’s instructions.

Forty-eight hours later, the full medium was replaced with the basal medium for later SD experiments.10,11 Apoptotic cells were determined by terminal deoxynucleotidyl transferase biotin dUTP nick end labelling (TUNEL) assay using an in situ cell death detection kit (Roche Diagnostic, Indianpolis, IN) as described in our previous publications.25,26 The 3′-OH ends of fragmented nucleosomal DNA were specifically labelled in situ in the presence of Sclareol exogenously added terminal transferase biotin-labelled dUTP, NVP-BKM120 and were detected with alkaline-peroxidase-conjugated anti-fluorescein antibody. Cells were fixed on coverslips with ice cold 4% paraformaldehyde for 30 min and exposed for the appropriate time to a permeabilization solution (0·1% Triton X-100, 0·1% sodium citrate). Coverslips were coated with poly-d-lysine. After washing, 50 μl of TUNEL reaction mixture was placed on the cells and then incubated in a humidified atmosphere for 60 min at 37°. Fifty microlitres of substrate solution was added onto coverslips following convert-AP incubation. Finally

coverslips were washed with phosphate-buffered saline and mounted with citiflor. Apoptosis was quantified by scoring the percentage of cells with positive staining at the single cell level. Apoptotic versus total cells were counted in at least five randomly chosen microscopic fields (magnification 20 ×) and 1000 total cells. Western blot was performed as described previously.27 Briefly, protein was extracted by Lysis buffer containing 1% nonidet P-40, 50 mm HEPES, 150 mm NaCl, 1 mm ethylenediaminetetraacetic acid, 1 mm phenylmethylsulphonyl fluoride, 0·1% sodium dodecyl sulphate (SDS), 0·1% deoxycholate and 500 μm orthovanadate. Bradford method was used to determine protein concentration.