FIGURE 2.

Mutated constructs impair Rad9B both subcellular localization, DDR checkpoint and JNK activation functions. (a) HeLa cells were transfected with mutated and wildtype constructs for 24-hr incubation and then were imaged under a deconvolution microscope. FS1: Ser318FS; FS2: Arg401FS. (b) HeLa cells were transfected for 24 hr and followed with 45 min hydroxyurea treatment and then imaged under a fluorescent microscope. The right panel shows a zoomed-in wildtype cell forming strong foci signal. ɤ-H2AX, a biomarker for DNA double-strand break (c) Western blot assay showed a decreased expression level of FS1 and FS2 mutants in HEK293T overexpression model. The predicted size of the wildtype and other mutants RAD9B are 46 Kd, while the FS1 is predicted to be 35 Kd and FS2 is 44 Kd. In (c), FS1 showed a band approximately 35 Kd, which is consistent with prediction. FS2 showed an unclear band at 44 Kd. We assumed that the protein may have a degradation. (d) HEK293T cells were transfected with wildtype and different RAD9B mutants. JNK and phosphorylated JNK level are both examined by western blot. RAD9B mutant overexpression except for p.Phe215Leu and P.Gly221Arg show a significant decreased level of p-JNK activation. *p < .05. DDR, DNA damage response; n.s, not significant; WT, wildtype