Figure 2. Analysis of wild type HSF4b and mutant HSF4b^lop11 proteins. A: western blot of nuclear extracts transfected with wild type Hsf4b or Hsf4b^lop11 clones. B: EMSA showed absence of HSE-DNA binding of HSF4b^lop11 when compared to wild type HSF4b. A negative control was a binding reaction with nuclear extracts following transfection with an empty vector (pcDNA3.1; lanes 1 and 6). Labeled HSE and wild-type HSF4b (lane 2) form a complex (arrow). Specificity of HSF4b binding to HSE was determined by a specific competition with cold HSE (lane 3), nonspecific competition by addition of unlabeled SP1 (lane 4) and supershift (asterisk) by addition of His-antibody (lane 5). Nuclear extracts from cells transfected with Hsf4b^lop11 did not result in HSE-DNA binding in the presence of labeled HSE (lane 7), presence of labeled and unlabeled HSE (lane 8), presence of labeled HSE and unlabeled SP1 (lane 9) and labeled HSE and His-antibody (lane 10). C: Transactivation of HSE-reporter in HEK293 cells by wild type HSF4b and HSF4b^lop11 proteins. Luciferase values were normalized to β-galactosidase activity, averaged for three separate transfections and expressed relative to the ration for wild-type Hsf4b. Error bars represent SEM. Asterisk indicates samples with a significant difference (p<0.05; t-test) calculated from comparison with wild-type. D: western blot analysis of lens protein extracts from P7 wild type (wt) and lop11 lenses immunobloted with CRYAB-antibody (top panel) showing severely reduced CRYAB levels in lop11 lenses. The molecular mass is indicated to the right of each blot. Even loading was confirmed by immunobloting with β-actin (bottom panel).