Modulation of STAT3, but not NF-κB and PI-3 K and ERK/MEK, is involved in the inhibition of IL-6-induced IgM secretion by SR144528. (A) SKW 6.4 cells were pretreated with CB2 antagonist SR144528 (10 μM) or CB1 antagonist SR141716 (5 or 10 μM) or the indicated inhibitors (Bay11-7085, 0.01 μM; U0126: 15 μM; LY294002, 15 μM) for 30 min, followed by IL-6 (100 U/ml) exposure for 4 days. IgM in the culture supernatants was measured by ELISA. Background IgM secretion was 99 ng/ml and was subtracted from the IL-6-induced secreted IgM shown. (B) SKW 6.4 cells were pretreated with SR144528 (SR) or Bay11-7085 (Bay) for 30 min, followed by IL-6 (300 U/ml) stimulation for another 45 min. After harvest, the whole-cell lysates were subjected to Western blot analysis and detected with indicated antibodies. β-Actin served as loading control. The numbers below the IκB-α and p-STAT3 (705) bands indicated the normalized ratios of the target band and their respective β-Actin. (C-D) SKW 6.4 cells were treated with IL-6 in the absence or presence of SR144528 (SR) for 24, 48 or 96 hrs, respectively. Total cellular RNA was extracted with Trizol Reagent and reverse transcribed into cDNA by using SuperScript RT III enzyme. SYBR Green-based real-time PCR for each gene was performed and the results were normalized against the control of GAPDH signals. Final concentrations of DMSO in the tested wells were equal or less than 0.05%.