Fig. 2: Investigation of FIKK4.1 and FIKK4.2 local protein environment.

a, The subcellular localization of FIKK4.1 and FIKK4.2 investigated by immunofluorescence assay using anti-HA antibodies (magenta) targeting the C-terminally HA-tagged FIKK4.1 and anti-FIKK4.2 antibodies (green). DAPI (blue) is used for nuclear staining. Scale bars, 5 µm. The assay was performed three times with similar results. b, A diagram representing the proximity labelling workflow. FIKK4.1 (top) and FIKK4.2 (bottom) were tagged with a TurboID biotin ligase. Upon addition of biotin, proteins in the vicinity (represented by an orange area with a dashed outline) of the bait are biotinylated on lysine residues (represented by yellow stars). iRBCs were lysed in 8 M urea in 50 mM HEPES and proteins were trypsin digested into peptides. Biotinylated peptides were enriched using beads coated with two different anti-biotin antibodies and analysed by LC–MS/MS. c, A network analysis of FIKK4.1 and FIKK4.2::TurboID data. The connecting lines indicate a protein that is probably in the vicinity of the TurboID-tagged protein. Blue depicts proteins identified as potential FIKK4.1 direct targets in ref. ¹⁶. Red depicts proteins that have been identified as potential FIKK4.2 direct targets, and green depicts proteins identified as potential targets of both FIKK4.1 and FIKK4.2. The thickness of the connection represents how well the phosphorylation site matches the corresponding in vitro preferred phosphorylation motifs (of FIKK4.1 or FIKK4.2) from Supplementary Fig. 3.