Figure 2. Cyclin-F proximity and interaction proteomics.

(A, B) Lysates from empty and HA-FLAG-CCNF or -TBC1D7 (A) and APEX2-CCNF (B) overexpressing SH-SY5Y and N2a cells were analyzed by SDS–PAGE and immunoblotting. (C) Biotinlyation in SH-SY5Y and N2a cells overexpressing APEX2-CCNF was induced by 30 min biotin-phenol (BP) and 1 min H₂O₂ treatment followed by lysis and immunoblot analysis. (D) Schematic overview of proximity and interaction proteomics workflow. (E) Volcano plots showing changes in abundance of candidate interacting proteins of HA-FLAG-TBC1D7 (control) and HA-FLAG-CCNF in N2a and SH-SY5Y (upper panel) and of proximity partners of APEX2-CCNF in the absence (control) and presence of BP in N2a and SH-SY5Y (lower panel). Proteins enriched in HA-FLAG-CCNF immune complexes or in proximity to APEX2-CCNF are shown in orange (t test difference > 0.5, q-value < 0.05, FDR-corrected, t test). Top 10 significantly enriched proteins, known CCNF interactors (black circles) and selected candidates are highlighted. (F) Overlap between proximity partners (q-value < 0.05, t test difference > 2, t test) and interaction candidates (q-value < 0.05, t test difference > 1, t test). (E, G) Gene Ontology (GO) analysis of proteins found in the overlap of (E). Grey gradient represents P-values. The number of proteins (count) found associated with a given GO term is indicated by the size of the circles. BP, biological process; CC, cellular component; MF, molecular function.