Signaling molecules involved in responses to Cao2+. (A) Effects of NF-κB inhibitors on CD83 expression. (i) Isolated B cells were cultured for 1 hour with CAPE (25 μg/mL), Bay 11–7082 (1 μM), or wedelolactone (150 μM) prior to RNA extraction and RT-PCR. (ii) PBMCs were cultured for 4 hours prior to flow cytometric analysis of CD83 expression in B cells (CD19+-gated PBMCs). Baseline CD83 expression was analyzed immediately after cell isolation. (B) Isolated B cells were cultured in AIM-V for 0, 2, 5, 10, 30, and 60 minutes prior to lysis. Samples were then electrophoresed and immunoblotted for phosphorylated (P)–IκB, total IκB, P-AKT, P-ERK, P-p38 MAPK, P-serine PKC substrate, P-CaMKII, and β-actin (loading control). Similar results were obtained using samples from 3 different donors. (C-E) Cells were incubated in AIM-V with chlorpromazine (Chlor, 2 μM), CsA (1 μg/mL), KN-93 (20 μM), Gö6976 (1 μM), PD98059 (20 μM), U0126 (20 μM), SB203580 (2 μg/mL), Ly294002 (20 μM), or wortmannin (100 nM) for 4 hours. Statistical analysis was performed on percentage CD83 expression of treated compared with untreated (“alone”) samples (C) or of samples treated with a combination of inhibitors compared with samples treated with one inhibitor (D,E). Data were obtained by gating on CD19+ cells in PBMC populations. *P < .05; **P < .001. (F) Protein extracts were obtained from isolated B cells immediately after isolation (baseline) or after 10 minutes in culture in AIM-V alone or with LY294002, Gö6976, PD98059, KN-93, or its inactive analog KN-92, and then analyzed by immunoblotting. Similar data were obtained from 2 additional donors.
