INT6 interacts with SLBP. (A) Extracts of HeLa cells were used for immunoprecipitation experiments performed with pre-immune serum (p.i., lane 1), protein A-Sepharose beads only (lane 2), and the C-20 antibody to INT6 (lane 3). Immunoprecipitates were analyzed by immunoblot using a monoclonal antibody directed against SLBP (top panel) and an antibody to INT6 (bottom panel). Positions of the SLBP and INT6 signals are indicated on the right of the gel together with the position of bands of a molecular weight marker run in parallel. (B) HeLa cells were transfected with the pTL-INT6 (lanes 1 and 2) and pSG-FLAGSLBP (lane 2) expression vectors. Lysates from these transfected cells were analyzed by immunoblot using an antibody to INT6 (top panel) and to Flag (second panel from top). With these cell extracts, immunoprecipitation was performed using the antibody to INT6 (C20) in the presence of RNAse A, and the immunoprecipitates were analyzed by immunoblot with the monoclonal antibody to Flag (third panel from top) and to INT6 (bottom panel). The position of FLAGSLBP is indicated. (C) HeLa cells were transfected with control (lane 1), MIF4GD (lane 2), or EIF3B (lane 3) siRNAs. Extracts of these cells were analyzed with antibodies to MIF4GD (top panel), to EIF3B (middle panel), and to β-actin (bottom panel). (D) These extracts were used for immunoprecipitation experiments performed using the C-20 antibody to INT6, and immunoprecipitates were analyzed with the antibody directed to SLBP (top panel) as in A and with the antibody to INT6 (bottom panel).
