Skip to main content
. Author manuscript; available in PMC: 2021 May 28.
Published in final edited form as: Nat Cell Biol. 2021 Apr 1;23(4):341–354. doi: 10.1038/s41556-021-00653-6

Extended Data Fig. 3. EZH2 bridges FBL-NOP56 interaction by binding to both proteins.

Extended Data Fig. 3

(a) Western blot analysis of FBL protein level in PCa cell lines upon EZH2 knockdown.

(b) Western blot analysis of EZH2 and H3K27me3 levels in PCa cell lines upon FBL knockdown.

(c) Equal amount of FBL protein in control and EZH2-deficient C4–2 cells were pulled down using anti-FBL antibody followed by western blot to detect its trimethyl-lysine (Kme3) level.

(d) Co-IP of Nop56, Nop58 and Snu13 with Fbl in control and Ezh1/Ezh2 double-knockout XEN cells, followed by western blot analysis with indicated antibodies. Graph represents the relative Nop56 protein level coimmunoprecipitated with Fbl in each group. Data represent Mean ± SD (n=3 biologically independent measurements). Interaction intensity at control XEN group was set as 1. Statistical significance was determined by two-tailed Student’s t-test.

(e) AlphaLISA cross-titration assay to determine the optimal protein concentration combination of FBL and NOP56. A hook point is reached at 3 nM His-tagged NOP56 and 30 nM Flag-tagged FBL. To achieve best results, a combination of 3 nM His-tagged NOP56 and 3 nM Flag-tagged FBL was used for the subsequent experiments.

(f) AlphaLISA displacement assay showing that FBL-NOP56 interaction is unaffected by EED. Data represent Mean ± SD for n=3 biologically independent experiments.

(g) After nucleolar isolation, proteins in each fraction were separated by SDS-PAGE and visualized by UV (upper panel). Distributions of EZH2, FBL (nucleolar marker), NOP56, FUS/TLS (nucleoplasmic marker) and β-actin (cytoplasmic marker) in each fraction were detected by western blot (lower panel). Wc: whole cells; CN: cytoplasm + nucleoplasm; No: nucleoli.

(h) AlphaLISA cross-titration assay to determine the optimal protein concentration combination of Flag-tagged-EZH2 and GST-tagged-NOP56. A hook point is reached at 100 nM Flag-tagged-EZH2 and 1 nM GST-tagged-NOP56.

(i) Co-IP of Myc-tagged FBL with full-length or truncation mutants of Flag-tagged NOP56.

The assays in a-c, e and g-i have been performed three times with similar results. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 3.