Phospho-Chk2 (Thr68) Antibody - #AF3036

FIGURE 7 Fbxw24 knockout increased RAD51 and p-CHK2 foci in female germ cells. (A and B) Immunofluorescence and quantification showed that RAD51 foci significantly increased in the Fbxw24-KO zygotene or pachytene germ cells. DNA in blue, RAD51 in green, and SYCP3 in red. (C and D) Western blots and quantification show that the RAD51 level significantly increased in the Fbxw24-KO 16.5 DPC genital ridge. (E) RAD51 antibody immunoprecipitation and UB46 western blots demonstrate that RAD51 can be ubiquitinated, while Fbxw24 knockout substantially reduced RAD51 ubiquitination level. Further, exogenous FBXW24 protein (1 μg) supplementation increased RAD51 ubiquitination level and reduced RAD51 level close to WT. (F and G) Immunofluorescence and quantification showed that the p-CHK2 foci significantly increased in the Fbxw24-KO zygotene or pachytene germ cells. DNA in blue, p-CHK2 in green, SYCP3 in red. (H and I) Western blots and quantification show that the p-CHK2 level significantly increased in the Fbxw24-KO 16.5 DPC genital ridge. (J) p-CHK2 antibody immunoprecipitation and UB46 western blots demonstrate that RAD51 can be ubiquitinated, while Fbxw24 knockout substantially reduced p-CHK2 ubiquitination level. Further, exogenous FBXW24 protein (1μg) supplementation increased p-CHK2 ubiquitination level and reduced RAD51 level close to WT. α-tubulin or GAPDH was used as a loading control. *, p < 0.05; **, p < 0.01. AU, arbitrary unit

FIGURE 7 Fbxw24 knockout increased RAD51 and p-CHK2 foci in female germ cells. (A and B) Immunofluorescence and quantification showed that RAD51 foci significantly increased in the Fbxw24-KO zygotene or pachytene germ cells. DNA in blue, RAD51 in green, and SYCP3 in red. (C and D) Western blots and quantification show that the RAD51 level significantly increased in the Fbxw24-KO 16.5 DPC genital ridge. (E) RAD51 antibody immunoprecipitation and UB46 western blots demonstrate that RAD51 can be ubiquitinated, while Fbxw24 knockout substantially reduced RAD51 ubiquitination level. Further, exogenous FBXW24 protein (1 μg) supplementation increased RAD51 ubiquitination level and reduced RAD51 level close to WT. (F and G) Immunofluorescence and quantification showed that the p-CHK2 foci significantly increased in the Fbxw24-KO zygotene or pachytene germ cells. DNA in blue, p-CHK2 in green, SYCP3 in red. (H and I) Western blots and quantification show that the p-CHK2 level significantly increased in the Fbxw24-KO 16.5 DPC genital ridge. (J) p-CHK2 antibody immunoprecipitation and UB46 western blots demonstrate that RAD51 can be ubiquitinated, while Fbxw24 knockout substantially reduced p-CHK2 ubiquitination level. Further, exogenous FBXW24 protein (1μg) supplementation increased p-CHK2 ubiquitination level and reduced RAD51 level close to WT. α-tubulin or GAPDH was used as a loading control. *, p < 0.05; **, p < 0.01. AU, arbitrary unit

Fig. 4. Changes of ATM and its downstream proteins in GC-1 cells and mouse testis. Expression and analysis of the related proteins in mouse testis (A) and in GC-1 cells (B). *p < 0.05 vs. the control group; #p < 0.05 vs. the corresponding MC-LR exposure group. All data were expressed as  ± SD (n = 3).

Figure 4.| - p21 knockdown reduces the DNA damage response in HK-2 cells. (A) Protein expression levels of p-ATM, ATM, p-Chk2, Chk2, rH2AX, p-p53 and p53 were analyzed using western blotting in cells transfected with shp21 or shCon with or without AA-induced injury.

Figure 3. Effects of AA on the meiotic process of pachytene spermatocytes and the expression of meiotic DSB signaling proteins. (a) Representative immunofluorescence images of SYCP3 and γH2AX staining in the pachytene spermatocytes of the testis. (b) and (c) The expression levels of meiotic DSB signaling proteins (γH2AX, p-ATM and p-CHK2) in the testis and isolated spermatocytes, respectively. The data are expressed as the mean ± SD of three mice in each group for annotation B and expressed as the mean ± SE of three separate experiments with triplicate samples for annotation C. *p < 0.05, **p < 0.01, compared with the control group.