Extended Data Fig. 1: EB1 interacts with and is crotonylated by TIP60.
From: Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis
(a) HEK293T cells co-transfected with FLAG-EB1 and GFP, GFP-PCAF, GFP-TIP60 or myc-p300. 24 hours after transfection, FLAG-EB1 was immuno-purified and subject to Western blotting analyses, and crotonylation of EB1 was judged by anti-pan-CrK antibody. (b) In vitro TIP60 acetylation/crotonylation reactions were performed with crotonyl-CoA (Cr-CoA) and acetyl-CoA (Ac-CoA). Reaction products were immunoblotted with the indicated antibodies. FLAG-TIP60 was purified from HEK293T cells. Aurora B-mCherry-His and p53-mCherry-His were purified from E. coli and used as substrates. The acetylation and crotonylation level were detected with an anti-pan-AcK and anti-pan-CrK antibody. Protein levels were analyzed with SDS-PAGE gel stained by CBB. (c) HEK293T cells were co-transfected with FLAG-EB1 and GFP or GFP-TIP60 for 24 h, and their interactions were tested by co-immunoprecipitation. Western blotting analyses were conducted by anti-GFP and FLAG. (d) HEK293T cells were co-transfected with FLAG-TIP60 and GFP or EB1-GFP for 24 h, and their interactions were tested by co-immunoprecipitation. Western blotting analyses were conducted by anti-GFP and FLAG antibodies. (e) Schematic illustration of EB1 structural domains and truncation mutants. Their binding properties with TIP60 were also annotated. (f) Bacterially expressed GST-EB1 full-length (FL) and truncation mutants were purified and used as affinity matrices to isolate His-TIP60. The binding fraction was analyzed by immunoblotting and protein levels were analyzed with SDS-PAGE gel stained by CBB. An asterisk indicates the band corresponding to the protein of interest, if multiple bands were observed. (g) Schematic illustration of TIP60 structural domains and truncation mutants. (h) Bacterially expressed GST-EB1 was purified and used as affinity matrix to isolate GFP-TIP60 full-length (FL) or truncation mutants expressed in HEK293T cells. Binding activity was analyzed by immunoblotting with an anti-GFP antibody. (i) The FLAG-EB1 proteins from Fig. 1a were analyzed by mass spectrometry. The typical EB1 Lys66 crotonylation mass spectrum was shown. Related to Fig. 1.
