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. 2018 Sep 14;37(20):e99395. doi: 10.15252/embj.201899395

Figure EV6. CDK1‐driven BCL9 phosphorylation at T172 regulates BCL9 interaction with clathrin in mitosis (related to Fig 6).

Figure EV6

  1. Immunoblotting analysis of BCL9 T172 phosphorylation in mitotic cells with BCL9MF or T172AMF expression by two anti‐P‐T172‐BCL9 rabbit polyclonal antibodies.
  2. Immunoblotting analysis of endogenous BCL9 T172 phosphorylation in mitotic cells by a selected anti‐P‐T172‐BCL9 rabbit polyclonal antibody.
  3. Immunohistochemical staining of P‐T172‐BCL9 in a human HCC tissue. Scale bars represent 50 μm.
  4. Immunofluorescence analysis of P‐T172‐BCL9 subcellular localisation in mitotic cells treated with or without P‐T172 peptide blocking. Scale bars represent 10 μm.
  5. ELISA analysis to confirm anti‐P‐T172‐BCL9 antibody recognising T172 phosphorylation peptide specifically.
  6. Immunoblotting analysis of the input for Fig 6D.
  7. CHX chase analysis of the stability of overexpressed or endogenous proteins in mitotic cells with BCL9 stable knockdown by shBCL9‐UTR.
  8. Quantification of protein stability in the CHX chase assay mentioned above.
  9. Immunoblotting analysis of the BCL9 and its mutants expression in shBCL9‐UTR knockdown cells.
  10. Live‐cell imaging analysis of cell division problem by stable expression of BCL9, T172A or T172D. Twenty‐three H2BGFP‐mitotic stable cells were monitored for each time experiment; data are shown from three independent experiments. Significance was measured by two‐tailed unpaired t‐test, *P < 0.05, **P < 0.01, ***P < 0.001. All data are the mean ± SD.
  11. Oncogenic soft agar colony formation assay of cells stably expressing BCL9, T172A or T172D.

Source data are available online for this figure.