Fig. 5

FBXO22 promotes degradation of BACH1 in MLLr AML cells. A The workflow for identifying FBXO22-interacting proteins in THP-1 cells by LC-MS/MS and the heat map analysis of 28 overlapping proteins identified in two independent experiments. B Western blot analysis of indicated proteins in the inputs and immunoprecipitates of Flag-tagged FBXO22 transfected THP-1 cells. C Western blot analysis of indicated proteins in the inputs and immunoprecipitates of endogenous BACH1 in THP-1 cells. D Western blot analysis of indicated proteins in three clonally derived THP-1 cell lines depleted of FBXO22 (gFBXO22#1, gFBXO22#2 and gFBXO22#3) with three negative controls (gNC). E Western blot analysis of indicated proteins in THP-1 and MV4-11 cells infected with shNC, shFBXO22#1 and/or shFBXO22#2. The asterisk represents a nonspecific band. F Western blot analysis of indicated proteins in MLL-AF9⁺ AML BM cells from primary recipients injected with Fbxo22^+/+, Fbxo22^−/−, Scl-Cre⁺;Fbxo22^fl/fl or Scl-Cre⁻;Fbxo22^fl/fl AML cells. G Western blot analysis of indicated proteins in THP-1 cells infected with EV and Flag-tagged FBXO22. H A Dox-inducible expression system encoding FBXO22 was introduced by lentiviruses into THP-1. Western blot analysis of indicated proteins in THP-1 cells by Dox administration for indicated times. I Western blot analysis of indicated proteins of THP-1 gNC#1 and gFBXO22#3 cell lines treated with CHX (100 μg/ml) for indicated times. J Pearson’s correlation between FBXO22 and BACH1 protein expression level of AML patients from PDX032110 database. K BACH1 protein expression level was compared between FBXO22^High and FBXO22^Low protein expression groups in PDX032110 database. Categorization of samples into FBXO22^High and FBXO22^Low protein expression was determined by maximally selected rank statistics method. L Western blot analysis of indicated proteins in THP-1 gFBXO22#3 cells infected with EV and Flag-tagged FBXO22 and treated with MG132 (10 μM), MLN4924 (10 μM), BFA (10 μM) and NH₄Cl (10 mM) for 24 h. M, N THP-1 gFBXO22#3 cells infected with EV and Flag-tagged FBXO22 (M), THP-1 gNC#1 and gFBXO22#3 cells (N) were treated with 10 μM MG132 for 6 h, harvested, and submitted to in vivo ubiquitination assay, followed by Western blot analysis with indicated antibodies. Error bars denote mean ± SD. Statistical significance was determined by two-tailed unpaired t test (K) and the P values were shown. All experiments were repeated at least three times with similar results. The numbers in D–I and L show the quantification of protein levels