Fig. 1

TMEM55B interacts with members of the NEDD4-like family of E3 ligases.

a Schematic of the predicted TMEM55B membrane topology. Illustration created with BioRender.com. b Multi-sequence alignment of TMEM55B orthologs in different species. The PPXY motif is indicated in red. c U2OS cells were transfected with plasmids encoding TMEM55B-GFP-WT full length (FL), TMEM55B-GFP-WT cytosolic domain (CD), TMEM55B-GFP-P66A FL, or P66A CD. Cells were lysed and pulled down with GFP beads. The results are representative of three independent experiments. WT (Wild-type), P66A (P66A mutant). d U2OS TMEM55B KO cells stably expressing mCherry-NEDD4 (red) were transfected with plasmids encoding GFP, TMEM55B-GFP-WT or P66A (green). Cells were fixed and permeabilized for immunofluorescence. Scale bars, 20 μm, n = 3. e U2OS cells were infected with adenovirus expressing TMEM55B-GFP-WT or P66A and pulled down with GFP beads. The results are representative of three independent experiments. f U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT were treated with various drugs and pulled down with GFP beads. EBSS for 4 h, NaAsO₂ (300 μM) for 2 h, H₂O₂ (500 μM) for 4 h, LLOMe (1 mM) for 2 h, CCCP (25 μM) for 4 h, Tunicamycin (10 μg/ml) for 4 h, Thapsigargin (10 μM) for 4 h. The results are representative of three independent experiments. g U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A (green) were treated with or without NaAsO₂ (300 μM) for 2 h. Cells were fixed and immunostained with antibodies against ubiquitin (red) and LAMTOR1 (blue). Scale bars, 20 μm, n = 3. h U2OS cells were transfected with plasmids encoding TMEM55B-GFP-WT or P66A and treated with or without NaAsO₂ (300 μM) for 2 h. Cells were lysed and immunoprecipitated with GFP beads under denaturing condition. The results are representative of three independent experiments. Source data are provided as a Source Data file.