Fig. 1

Determination of the stimulation concentration and time of tunicamycin inducing ER stress. (A) The protein expression of GRP-78 and GAPDH when the IPEC-J2 cells were stimulated for 6 h with different concentrations of tunicamycin (0, 0.5, 1, and 2 µg/mL). The blots were cut prior to hybridization with antibodies in order to focus on the specific protein bands of interest. As a result, we do not have full-length images of the membranes. However, the cropped images accurately reflect the expression of the target protein. (B) The relative gray value of GRP-78 when the IPEC-J2 cells were stimulated for 6 h with different concentrations of tunicamycin (0, 0.5, 1, and 2 µg/mL). (C) The protein expression of GRP-78 and GAPDH when the IPEC-J2 cells were stimulated for different durations (0, 6, 9, 12, and 15 h) with 1 µg/mL tunicamycin. (D) The relative gray value of GRP-78 when the IPEC-J2 cells were stimulated for different durations (0, 6, 9, 12, and 15 h) with 1 µg/mL tunicamycin. (E) Apoptosis presentation using AO/EB fluorescent staining when the cells were stimulated for 9 h with 1 µg/mL tunicamycin to build the ER stress model. The cell morphology was photographed with an inverted microscope (×200). The viable cells appeared uniformly green, the apoptotic cells showed bright green in the nuclei as chromatin condensation and nuclear fragmentation, and the necrotic cells presented bright orange. Different lowercase letters on the graph bars indicate statistically significant differences among the groups (ANOVA with Duncan’s test, p < 0.05). Data are presented as means ± SEM. The blots were cropped. The samples derived from the same experiment and that blots were processed in parallel. Tuni, tunicamycin; GRP-78, glucose-regulated protein 78; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, an internal reference protein