Fig. 3.

Noncanonical NF-κB binds to the promoter region of ICOSL gene and is critical for ICOSL induction. (A) Spleen B cells from WT mice were either not treated (NT) or stimulated with BAFF for 24 h. Chromatin IP was performed using either a control Ig (Ig) or anti-RelB antibody, and the precipitated DNA was subjected to PCR using primers that amplify the indicated regions of the ICOSL promoter. Input DNAs also were subjected to PCR to show the efficiency of the primers. (B) WT spleen B cells (Left) or M12 B cells (Right) were stimulated with BAFF for the indicated times. Chromatin IP was performed using either a control Ig or the indicated antibodies, and the precipitated DNA was subjected to PCR using primers that amplify a 300-bp DNA fragment (−490 to −190) of the ICOSL promoter. Data are representative of three independent experiments. (C and D) M12 cells were infected with either the pLKO.1 lentiviral vector or the same vector encoding RelB shRNA. After puromycin selection, the bulk of infected cells were subjected to IB to determine the efficiency of RelB knockdown (C). The control and RelB knockdown cells were either not treated (NT) or stimulated with BAFF for 48 h, and the ICOSL expression level was analyzed by flow cytometry (D). (E) WT or NIK KO splenocytes were infected with retroviruses carrying the pCLXSN(GFP) vector (vector), or pCLXSN(GFP)-NIK (NIK). Infected cells were stimulated with BAFF for 48 h, and ICOSL expression on infected B cells was analyzed by flow cytometry (gated on B220⁺GFP⁺ cells).