The intracellular FasL domain binds to Lef-1 and influences its transcriptional activity. (A) In vitro GST pull-down experiments reveal FasL-Lef1 interaction. Top panel, pull-down of in vitro translated ³⁵ [S] FasL (left), FasL ICD (middle), and Lef-1 (right) proteins with the indicated bacterially expressed GST fusion proteins. The black arrows mark in vitro translated proteins that specifically interact with the GST fusion molecules. GST-PSTPIP serves as a positive control for FasL binding¹³  and GST protein only as a general negative control. Bottom panel, Coomassie-stained protein gels showing purified GST fusion proteins. Arrows indicate the GST fusion proteins used for the above GST pull-down experiment. (B) Coimmunoprecipitation of overexpressed Flag-FasL and Lef-1 proteins. HEK 293T cells were transfected with Lef-1 and FasL or empty vector (pcDNA3.1). Forty-eight hours later, cell lysates were prepared and immunoprecipitations performed with anti-Flag M2 (binding to Flag-tagged FasL) or anti-FasL (NOK1) antibodies. Western Blot analysis with the anti–Lef-1 antibody revealed for both coimmunoprecipitation reactions binding of Flag-FasL to Lef-1 (top panel). Control incubation of the membrane with anti-Flag antibody confirmed the presence and successful immunoprecipitation of Flag-FasL (middle panel right side); the input Western Blot analysis with anti–Lef-1 antibody (bottom panel) demonstrated presence of transfected Lef-1. (C) The FasL ICD represses Lef-1/β-catenin activity in a Luciferase reporter assay. HEK293T cells were cotransfected with the indicated increasing amounts of the FasL ICD together with optimal (TOP) or mutated (FOP, negative control) Firefly luciferase reporter constructs for Lef-1 activity. Sixteen hours after cultivation of the cells in the presence of 30mM LiCl to inhibit GSK3β, Lef-1–dependent Luciferase activity was quantified in the cell lysates. Firefly luciferase activity was normalized to Renilla luciferase activity, and the value obtained for TOP plasmid without FasL ICD was set to 1. All samples were measured in triplicates. Control transfections with TOP plasmid alone without LiCl did not induce luciferase activity (data not shown). The columns represent the mean of 3 individual experiments, and error bars indicate the standard deviation. Statistical significance was assessed using Student t test (*P < .5; **P < .01; ***P < .001). (D) RNA and protein expression analysis of Lef-1 in mature B cells. Top panel, RT-PCR experiment with intron-spanning Lef-1–specific PCR primers and total RNA isolated from wild-type and FasL ΔIntra splenic B cells that were stimulated with 1 μg/mL anti-IgM antibody for 4 hours. RNA prepared from Lef-1–transfected HEK 293T cells served as a positive control, while water without RNA template was used as a negative control. Bottom panel, histogram of intracellular FACS staining confirms expression of Lef-1 in B cells. Naive splenic B cells were fixed, permeabilized, and incubated subsequently with anti–Lef-1 and secondary antibody. The solid curve represents wild-type cells, the dotted curve represents the FasL ΔIntra B cells, and the filled gray curve depicts the isotype control. One representative experiment of 6 is displayed.