Figure 3.
FAM134B is acetylated by CBP at K160. (A) The interaction of FAM134B with lysine acetyltransferase CBP. HA-tagged lysine acetyltransferases CBP, p300, GCN5, PCAF, TIP60, and ATAT1 were expressed individually in HEK293T cells, which simultaneously expressed FAM134B-Flag. IP was performed with anti-HA beads, which was followed by Western blot for FAM134B-Flag. The experiments were performed twice. (B and C) Endogenous CBP was translocated from nucleus to ER labeled by BAP31 upon Tg treatment (1 μM, 1 h). Scale bars, 10 µm. The colocalization was analyzed by Pearson’s correlation coefficient in C (n = 20 cells per group). Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (D and E) CBP was translocated from nucleus to cytoplasm under Tg treatment. HEK293T cells treated with Tg (1 μM) for 0, 0.5, 1 h were performed with nuclear and cytoplasmic protein extraction assay. Nucleus was labeled with Lamin-B1. Cytoplasm was labeled with Tubulin. The indicated proteins were detected by Western blot. Quantification of CBP is shown in E (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. *P < 0.05, **P < 0.01. (F) FAM134B K160 was acetylated by CBP in vitro. Recombinant protein FAM134B WT purified from E. coli was incubated with lysine acetyltransferase HA-tagged CBP, p300, TIP60, GCN5, PCAF purified from HEK293T cells in HAT buffer at 30°C for 3 h. FAM134B acetylation was analyzed by Western blot. (G) FAM134B K160 was acetylated by CBP in vitro. Recombinant protein FAM134B WT or FAM134B K160R purified from E. coil was incubated with HA-CBP purified from HEK293T cells in HAT buffer at 30°C for 3 h. FAM134B acetylation was analyzed by Western blot. (H) FAM134B K160 acetylation by CBP was abolished by CBP/p300 inhibitor C646 in vitro. Recombinant protein FAM134B WT or FAM134B K160R purified from E. coli was incubated with HA-CBP purified from HEK293T cells in HAT buffer in the presence or absence of C646 at 30°C for 3 h. FAM134B acetylation was analyzed by Western blot. (I) CBP KD downregulated K160 acetylation. HEK293T cells were transfected with control, shRNA1, shRNA2, shRNA3 targeting CBP. K160 acetylation, FAM134B, and CBP were detected by Western blot. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F3.

FAM134B is acetylated by CBP at K160. (A) The interaction of FAM134B with lysine acetyltransferase CBP. HA-tagged lysine acetyltransferases CBP, p300, GCN5, PCAF, TIP60, and ATAT1 were expressed individually in HEK293T cells, which simultaneously expressed FAM134B-Flag. IP was performed with anti-HA beads, which was followed by Western blot for FAM134B-Flag. The experiments were performed twice. (B and C) Endogenous CBP was translocated from nucleus to ER labeled by BAP31 upon Tg treatment (1 μM, 1 h). Scale bars, 10 µm. The colocalization was analyzed by Pearson’s correlation coefficient in C (n = 20 cells per group). Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (D and E) CBP was translocated from nucleus to cytoplasm under Tg treatment. HEK293T cells treated with Tg (1 μM) for 0, 0.5, 1 h were performed with nuclear and cytoplasmic protein extraction assay. Nucleus was labeled with Lamin-B1. Cytoplasm was labeled with Tubulin. The indicated proteins were detected by Western blot. Quantification of CBP is shown in E (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. *P < 0.05, **P < 0.01. (F) FAM134B K160 was acetylated by CBP in vitro. Recombinant protein FAM134B WT purified from E. coli was incubated with lysine acetyltransferase HA-tagged CBP, p300, TIP60, GCN5, PCAF purified from HEK293T cells in HAT buffer at 30°C for 3 h. FAM134B acetylation was analyzed by Western blot. (G) FAM134B K160 was acetylated by CBP in vitro. Recombinant protein FAM134B WT or FAM134B K160R purified from E. coil was incubated with HA-CBP purified from HEK293T cells in HAT buffer at 30°C for 3 h. FAM134B acetylation was analyzed by Western blot. (H) FAM134B K160 acetylation by CBP was abolished by CBP/p300 inhibitor C646 in vitro. Recombinant protein FAM134B WT or FAM134B K160R purified from E. coli was incubated with HA-CBP purified from HEK293T cells in HAT buffer in the presence or absence of C646 at 30°C for 3 h. FAM134B acetylation was analyzed by Western blot. (I) CBP KD downregulated K160 acetylation. HEK293T cells were transfected with control, shRNA1, shRNA2, shRNA3 targeting CBP. K160 acetylation, FAM134B, and CBP were detected by Western blot. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F3.

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