Fig 1: AST2 is required to regulate the CLK-PlBMAL1 heterodimer formation.A) The relative expression levels of AST2 in the brain at 03:00, 06:00, 12:00, and 21:00 (n = 9) after partial knock down of AST2 (grey) in the brain by dsRNA injection and detection by qPCR; dsGFP injection was used as a control (white). B) The relative expression levels of AST2 in hemocytes at 03:00, 06:00, 12:00, and 21:00 (n = 9) after partial knock down of AST2 (grey) by dsRNA injection and detection by qPCR; dsGFP injection was used as a control (white). Graph A and B represent AST2 mRNA levels, using qPCR. Results are representative of three independent experiments. Error bars indicate SD of nine replicates. The asterisks indicate significant differences (*P<0.05, **P<0.01); Student's paired t-test. C) The effect of dsAST2 on complex formation in the brain in vivo was examined by western blotting of brain extracts and detection as follows: for the CLK-PlBMAL1 complex an anti-BMAL1 antibody, for the AST2- PlRACK1-PlBMAL1 complex an anti-BMAL1 antibody, and for the AST2-PlRACK1 an anti- AST2 antibody was used. The horizontal band at the top of the histogram indicates the light condition (white = light, black = dark). D) Relative amounts of CLK-PlBMAL1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against BMAL1. Grey bars = dsAST2, white bars = dsGFP. E) Relative amounts of AST2-PlRACK1-PlBMAL1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against BMAL1. Grey bars = dsAST2, white bars = dsGFP. F) Relative amounts of AST2-PlRACK1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against RACK1. Grey bars = dsAST2, white bars = dsGFP. G) Relative amounts of AST2 protein in the brain in vivo was determined at different time points (n = 3) as determined by western blotting of brain extracts, using an antibody against AST2. Grey bars = dsAST2, white bars = dsGFP. Quantity One analysis was used to quantify the intensity of protein bands from three independent experiments and results are presented in graphs D to G. Statistical significance: *P<0.05, **P<0.01 using Student's paired t-test (error bars indicate SD from nine replicates).