Fig. 3: Kastor and Polluks are transmembrane polypeptides that localize to the OMM.

a Immunoblot analysis with antibodies to FLAG and to GAPDH (loading control) of HEK293T cells transiently transfected with the indicated vectors. b Immunofluorescence analysis of HeLa cells transiently expressing Kastor-(C)FLAG or Polluks-(C)FLAG with antibodies to FLAG. Mitochondria were detected with MitoTracker Red (Mito), and nuclei were stained with DAPI (blue). The boxed regions in the lower panels are shown at higher magnification in the corresponding upper panels. Scale bars, 10 μm. c Hydrophobicity scores of Kastor and Polluks determined by ProtScale. d Predicted transmembrane (TM) regions of human and mouse Kastor and Polluks analyzed by TMpred. e, f The mitochondrial fraction (Mito.) of HEK293T cells transiently expressing Kastor-(C)FLAG or Polluks-(C)FLAG (e) or of testis from Kastor^FLAG/+ and Polluks^FLAG/+ mice (f) was subjected to ultrasonic treatment, incubated with or without Na₂CO₃, and then centrifuged to yield a membrane fraction (ppt.) and a soluble fraction (sup.). These fractions were then subjected to immunoblot analysis with the indicated antibodies. g, h The mitochondrial fraction of HEK293T cells transiently expressing Kastor-(C)FLAG or Polluks-(C)FLAG (g) or of testis from Kastor^FLAG/+ and Polluks^FLAG/+ mice (h) was incubated in the absence or presence of proteinase K (ProK) and Triton X-100 and then subjected to immunoblot analysis with the indicated antibodies. Source data are provided as a Source data file.