Fig 1: The hATAC subunit KAT14 directly binds actin and favors binding to actin monomers via its C-terminus. (A) Schematic of the hATAC complex shown as a STRING map. Proteins indicated in the schematic have been described in Guelman et al. (2009). Proteins highlighted in blue are HCIs in our NLS-actin interactomes. ADA2A was observed only with NLS-actin AP-MS, and the other proteins are shared HCIs from NLS-actin AP-MS and NLS-actin BioID. (B) Western blots of the co-IP assay with 2Flag–KAT14 and 2HA–NLS-actin using HA (upper panel) or Flag beads (middle panel). Actin antibody (AC40) was used to visualize endogenous actin in a co-IP assay with or without overexpressed 2Flag–KAT14 and performed with anti-Flag antibody (lower panel). IP, immunoprecipitation sample. Molecular masses are indicated on the left. (C) Western blots of co-IP assays for 2Flag–KAT14 and the indicated HA-tagged actin mutants (upper panel). The lower panel shows the quantification of the relative amounts of actin mutants co-IP with KAT14. Data is normalized to wild-type actin, and is the mean±s.d. from three independent experiments. Dots in the graph represent individual data points from the independent experiments. P-values (*P<0.05) were determined with a one-sided Student's t-test showing significance between indicated samples: actin versus R62D-actin (P=0.04) and actin versus G168D, Y169D-actin (P=0.01). ns, not significant. (D) Western blots of co-IP assay performed for 2HA–actin and indicated 2Flag–KAT14 constructs. (E) Western blots of pulldown assay with actin, His–KAT14 and His–GST detected with the indicated antibodies. Note that for detecting the baits, unequal amounts were loaded on the SDS-PAGE gels (33% of the His–KAT14 sample and less than 1% of His–GST) for visualization purposes. See Fig. S4E for equal loading.
Fig 2: Actin inhibits KAT14 mediated H4K5 acetylation in vitro and in cells. (A) Western blots of an HAT assay with HeLa nuclear extract, and His-KAT14 without and with addition of actin and actin, probed with the indicated antibodies. –, no extract. (B) Quantification of the relative H4K5Ac abundance from western blots in A. Data is normalized to HeLa nuclear extract and is the mean±s.d. from three independent experiments. Dots in the graph represent individual data points from the independent experiments. P-values (*P<0.05) were determined with a two-sided Student's t-test showing significance between indicated samples: KAT14 versus KAT14+actin (P=0.02) and KAT14 versus negative control (–) (P=0.01). ns, not significant. (C) Western blots from total cell lysates of non-induced and tetracycline-induced stable HEK Flp-In cell lines expressing inducible HA-Strep–NLS-actin or HA-Strep–NLS-R62D-actin with the indicated antibodies. (D) Quantification of the relative H4K5Ac abundance from western blots in C. Data is normalized to the non-induced sample, and is mean±s.d. from four independent experiments. Dots in the graph represent individual data points from the independent experiments. P-values (*P<0.05) were determined with a one-sided Student's t-test showing significance between indicated samples (respectively, P=0.04 and P=0.01).
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