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. Author manuscript; available in PMC: 2021 Nov 5.
Published in final edited form as: Mol Cell. 2020 Oct 26;80(3):410–422.e6. doi: 10.1016/j.molcel.2020.10.008

Figure 5. Chk1 phosphorylates FAM122A at Ser37.

Figure 5.

(A) Western blots of the lysates from A549 cells treated with siControl or siCHK1, Samples were separated on a 12% Phos-tag-SDS PAGE gel. Phosphorylated and unphosphorylated FAM122A are labeled.

(B) Western blots of the lysates from 293T cells expressing Flag-tagged FAM122A (wild-type [WT] and S37A). Samples were separated on a normal SDS-PAGE gel (left) and a Phos-tag labeled SDS-PAGE gel (right). Phosphorylated and unphosphorylated FAM122A are labeled.

(C) Kinase assays of 293T cells expressing FLAG-FAM122A. 293T cells were transiently transfected with HA-Flag-tagged FAM122A, followed by immunoprecipitation with ant-Flag antibody. HA-Flag-FAM122A was incubated with ATP-γ-S and with/without purified CHK1. Phosphorylation of FAM122A was detected by thiophosphate.

(D) Anti-GST immunoprecipitation assays followed by western blots of stably expressing FAM122A-Flag 293T cells. FAM122A-Flag 293T cells were transiently transfected with PP2A-B55α-GST, and then treated with prexasertib at indicated concentrations for 24hrs, followed by GST-Pull down assay and western blots.

(E) Anti-FLAG immunoprecipitation assays followed by western blots of FAM122A-Flag 293T cells. Stably expressing FAM122A-Flag or control 293T cells were treated with siCHK1, followed by FLAG-Pull down assay, and western blots using anti-Flag, anti-PP2A-B55α, anti-14-3-3 and anti-H3 antibodies.

(F) Anti-FLAG immunoprecipitation (IP) assays followed by western blots of FAM122A-Flag 293T cells. 293T cells expressing Flag-tagged FAM122A (wild-type [WT] and S37A mutant) were used for Flag-IP and western blots.

(G) Survival plots of FAM122A-KO A549 cells transiently transfected with FAM122A-WT and -S37A mutant, followed by treatment with prexasertib for 3 days. Data are shown as mean ± SD from three independent experiments. sgFAM122A versus sgFAM122A+FAM122A, *P<0.05; sgFAM122A versus sgFAM122A+FAM122A-S37A, ***P<0.001, statistical analysis was performed using two-way ANOVA. The expression of the WT or mutant FAM122A protein is shown on the western blot (see inset).

(H) Illustration of the CHK1-FAM122A-PP2A-B55α pathway.