Over-expression of Alr4641/Alr4641C56S in
. (A, B) Fluorescence micrographs. The recombinant An4641+
(A) or AnC56S+
(B) cells were grown in BG-11 medium for 3 days and fluorescence microphotographs (500X magnification) using Hg-Arc lamp (excitation BP, 450–490 nm and emission LP, 515 nm) were captured. (C, D, E) Over-production of the Alr4641/Alr4641C56S protein in Anabaena. Cell-free extracts from the wild-type Anabaena PCC7120 (WT) or An4641+ or AnC56S+ (20 μg per lane) were resolved by reducing SDS-PAGE (C) or non-reducing SDS-PAGE (D) or by native polyacrylamide gel electrophoresis (E). After electrophoresis, proteins were immunodetected with the Alr4641 antiserum. The Alr4641 protein is indicated by an arrow. (F) Physical interaction of Alr4641 with NTRC. NiNTA agarose loaded with the His-tagged NTRC protein was incubated with cell-free extract obtained from An4641+ (+). Unloaded NiNTA agarose (i.e. free of any bound protein) was incubated with An4641+ cell-free extract as negative control (−). Bound proteins were resolved on SDS-Polyacrylamide gels, transferred onto nitrocellulose membrane and probed with the anti Alr4641 antibody. Input An4641+ cell extract added to NiNTA agarose containing NTRC (+In) or to the negative control (−In) is also shown. (G) Intracellular ROS formation in response to H2O2. WT or An4641+ or cells AnC56S+ were grown for 3 days in BG-11 medium and treated with H2O2 (1 mM) for 1 h. Subsequently, cells were incubated with DCHFDA (10 μM final concentration) for 20 min and fluorescence emission (λex = 490 nm, λem = 520 nm) was measured with a spectrofluorimeter. The relative fluorescence of control (untreated cells) and H2O2-treated cultures is shown in the figure.