2.12. Western Blotting

Mouse kidneys and HK-2 cells were lysed with cold lysis buffer (0.25 M NaCl, 50 mM Tris-HCl pH 7.4, 0.5% NP 40, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 1% cocktail, and 1 mM PMSF). BCA reagent was used to determine the total protein concentration. The separated proteins were then electrophoretically transferred to a polyvinylidene fluoride membrane. After blocking with 5% fat-free milk in PBST for 2 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C. After being washed three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 60 min. Finally, an ECL substrate (sc2048, Santa Cruz) was used to visualize the immunoreactive bands. The main antibodies used for Western blot analysis included PLK3 (4896S, 1 : 1000, Cell Signaling), PLK3 (DF4471, 1 : 1000, Afftiny), ATM (ab199726, 1 : 1000, Abcam), p-ATM (ab81292, 1 : 1000, Abcam), P53 (ab179477, 1 : 1000, Abcam), P53 (YT3528, 1 : 1000, Immunoway), p-P53 (9287S, 1 : 1000, Cell Signaling), rH2AX (YP0218,1 : 1000, Immunoway), Bcl-2 (BF9103, 1 : 1000, Affinity), and Bax (GB12-690, 1 : 1000, Servicebio); the relative expression of the targeted protein was normalized to that of β-actin (AC026, 1 : 5000, ABclonal).