ROCK1 interacts with p53 and regulates p53 levels in splenocytes. (A) PHZ was administered intraperitoneally (80 mg/kg) to WT and ROCK1^−/− mice. Spleens were harvested 4 days after PHZ injection and freshly isolated splenocytes were lysed in lysis buffer. Cell lysates were subjected to WB analysis using antibodies against p53 and caspase-3. Arrows show the expression of indicated proteins in WT and ROCK1^−/− spleen after PHZ treatment. Bottom panel shows β-actin levels in each lane (n = 3). (B) Freshly isolated splenocytes after PHZ injection were lysed in lysis buffer, and equal amount of lysates derived from untreated and PHZ-treated splenocytes were subjected to immunoprecipitation with an anti-p53 antibody. Western blot analysis was performed using an anti-ROCK1 antibody. Lanes 1 and 2 consist of lysates derived from untreated and PHZ-treated splenocytes from WT mice, respectively; lanes 3 and 4 consist of lysates derived from untreated and PHZ-treated splenocytes from ROCK1^−/− mice, respectively. The arrow indicates the interaction of ROCK1 with p53 in WT splenocytes. Total lysates were subjected to Western blot analysis with an anti-ROCK1 antibody. Lanes 5 and 6 consist of lysates derived from untreated and PHZ-treated splenocytes from WT mice, respectively; lanes 7 and 8 consist of lysates derived from untreated and PHZ-treated splenocytes from ROCK1^−/− mice, respectively. The arrow indicates the expression of ROCK1 in WT splenocytes (n = 3). (C) Splenocytes harvested after PHZ injection were lysed in lysis buffer, and equal amount of lysates derived from untreated and PHZ-treated spleens were subjected to Western blot analysis with an anti-phospho p53 (ser15) antibody. Lanes 1 and 2 consist of lysates derived from untreated and PHZ-treated splenocytes from WT mice, respectively; lanes 3 and 4 consist of lysates derived from untreated and PHZ-treated splenocytes from ROCK1^−/− mice, respectively. The arrow indicates the phospho-p53 (ser15) levels in WT and ROCK1^−/− splenocytes; middle panel arrow indicates total p53 levels in both genotypes, and bottom panel indicates β-actin controls in each line before and after PHZ stress. (D) Erythroid progenitor cells derived from the BM of WT and ROCK1^−/− mice were cultured for 7 days and treated with 50μM hydrogen peroxide or 2.5μM of caspase inhibitor for 1 hour. Cells were washed and resuspended in prewarmed PBS and loaded with 10μM 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) in the dark for 30 minutes at 37°C, 5% CO₂. Down arrow in each dot blot indicates percentage of ROS probe-positive cells in both genotypes. (E) Bar graph represents the mean ± SD of ROS levels in the WT and ROCK1^−/− erythrocytes (n = 6 mice per genotype, *P < .05).