Fig. 5.

IKKα controls AMBRA1 phosphorylation at S1014 during mitophagy. a Sequence alignment of the consensus sequence of IKKα-phosphorylation on IκBα, TAX1BP1 and AMBRA1. b HA-IKKα^K44M transfected HeLa cells were subjected to Co-IP and blotted for the indicated antibodies; n = 3. c Untransfected HeLa cells were treated with O/A (2.5 μM, 0.8 μM, 1 h) and with the IKKα irreversible inhibitor BAY-117082 (2 μM, 1 h); n = 3. d Representative image of HeLa cells transfected with PcDNA3, Flag-IKKα^WT or the kinase-dead HA-IKKα^K44M, treated with O/A and blotted for the indicated antibodies; n = 3. e In vitro kinase assay of immunoprecipitated-Flag-IKKα^WT- or HA-IKKα^K44M-expressing HeLa cells in which the substrate (Myc-AMBRA1^WT) was transcribed/translated in vitro. Samples were immunoblotted for the indicated proteins; n = 2. f In vitro kinase assay in which the immunoprecipitated fraction of IKKα^WT or IKKα^K44M were mixed to recombinant produced AMBRA1; n = 2. g HeLa cells transfected with Myc-AMBRA1^ActA or PcDNA3 (24 h) and treated with BAY-117082 inhibitor were blotted for the indicated antibodies; n = 3. h HeLa cells transfected with Myc-AMBRA1^WT or PcDNA3 and treated with O/A (2.5 μM, 0.8 μM, 5 h) and BAY-117082 inhibitor (2 μM, 5 h) were immunoblotted for the indicated proteins; n = 3. i HeLa cells transfected with PcDNA3 or HA-IKKα^K44M constructs in combination with Myc-AMBRA1^ActA were analysed by western blot for the indicated antibodies; n = 3. j PcDNA3+Myc-AMBRA1^WT or Myc-AMBRA1^WT+HA-IKKα^K44M co-transfected HeLa cells treated with O/A were subjected to immunoblotting for COXII, COXIV, HA, Myc and ACTIN antibodies; n = 3. Data represent the mean of three different samples (±S.D.) and are representative of experimental triplicate. *P < 0.05. Statistical analysis was performed using one-way ANOVA (c, g, h, I, j). M_r(K) = relative molecular mass expressed in kilodalton