Fig. 3. Cyclosporin A (CsA) does not alter activations of mitogen-activated protein kinases and protein kinase B (AKT) after H₂O₂ injury in human kidney proximal tubule epithelial cells. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H₂O₂ or control for 60 minutes (n=6 experiments per condition). (A) Phosphorylation levels of p38 (p-p38, 43 kDa), c-Jun N-terminal kinase (p-JNK, 46 and 55 kDa), and extracellular signal-regulated kinase (p-ERK, 42 and 44 kDa), and their total expression levels (t-p38, p-JNK, and p-ERK) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (B, C, E, F, H, I) Intensities of p-p38, t-p38, p-JNK, total JNK (t-JNK), p-ERK, and total ERK (t-ERK) protein bands were quantified using the AzureSpot software. (D, G, J) Activations of p38, JNK, and ERK indicated by their respective ratio of phosphorylation level to total expression level. (K) AKT phosphorylation (p-AKT) and total expression (t-AKT) with a molecular mass of 56 to 62 kDa were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (L, M) Intensities of p-AKT and t-AKT protein bands were quantified using the AzureSpot software. (N) AKT activation based on the ratio of phosphorylation to total expression. ^a)P<0.05 vs. control.