Preparation and verification of constructs and cell lines for proteomics studies. (A) Western blot analysis of whole-cell lysates (WCL) and immunoprecipitates from HeLa cells stably expressing ligase-trap constructs, showing that proteins were significantly enriched in α-FLAG IP. 2UF, 2×UBA-3×FLAG; 2US, 2×UBA-3×FLAG-SHKBP1. (B) Immunofluorescence (IF) of cell lines stably expressing ligase-trap constructs shown at low and high magnification. Cells were fixed, stained with α-FLAG (green) and DAPI (magenta), and imaged by confocal microscopy. Scale bars: 50 μm (first and third rows), 20 μm (second and fourth rows). (C) Western blot analysis of α-GFP IP from HeLa cells transfected with GFP-only or GFP-tagged versions of SHKBP1^WT or SHKBP1^F44A. (D) Confocal microscopy of HeLa cells stably expressing GFP-tagged SHKBP1^WT or SHKBP1^F44A. Scale bars: 50 μm (top row), 20 μm (bottom row). (E and F) Western blot analysis of co-IP experiments for HA-SHKBP1 and GFP-tagged hits from proteomics studies to demonstrate the interaction between SHKBP1 and other BTB-containing proteins (E) and substrate candidates (F). SHK, SHKBP1; KCT, KCTD3. Source data are available for this figure: SourceData FS1.