Figure 3
Detection of hepatic IP 3 Rs and RyR. The protein content of the calcium-release channels was evaluated by western blot (IP3Rs) and SDS-PAGE (RyR). Representative signals of IP3R1, IP3R2, and RyR and loading control (actin), and the daily rhythm profiles are shown in panels A, C, and E, respectively. Panels B, D, and F compare control groups of feeding condition (Fasted and Refed) for expression of the IP3R1, IP3R2, and RyR, respectively. Meanwhile IP3R1 and 2 were determined by conventional western blot, RyR protein was detected and quantified by stains-all methodology as described previously. Mean values for at least 4 independent experiments are shown. For IP3R type 1 and RyR proteins, 100 μg of the ER fraction was used. The plasma membrane fraction was used for IP3R type 2 (100 μg). Black circles correspond to AL and white circles to RF group. The light gray rectangle above x-axis indicates mealtime for the food restricted group (ZT4-ZT6). Mean values for at least 4 independent experiments are shown. Each data point was measured in triplicate. * (p < 0.05) significant difference between AL time points and + (p < 0.05) significant difference in the RF group between their time points (1-way ANOVA); # (p < 0.05) significant between AL vs RF (2-way ANOVA). ♦ (p < 0.05) significant difference between Fasted and Refed groups; x (p < 0.05) significant difference between Fasted and RF-ZT3; and & (p < 0.05) significant difference between Refed and RF-ZT6 (Student´s t-test).