Fig 1: Biochemical validation of candidate binding proteins detects PTK7 as the most robust GPR133 interactor(A) Validation affinity co-purification assay testing six of the top interactors identified in the screen with a TwinStrep-tagged GPR133-H543R bait in HEK293T cells. All candidate interactors were C-terminally tagged with the Myc epitope. (Ai) Western blots of input whole-cell lysates stained against Myc tag of ligand candidate proteins and anti-GPR133 C terminus. (Aii) Western blots of eluates after Strep-Tactin purification. Note that PTK7, and to a lesser extent TFRC, co-purify with GPR133. A representative blot of three biological repeats is depicted.(B) Membrane topology of PTK7. Note that the juxtamembrane portion of PTK7 is cleaved by MT1-MMP (membrane type 1 matrix metalloproteinase) and ADAMs (a disintegrin and metalloproteinases).(C) PTK7 is identified as one of the top interactors in the enrichment volcano plot as depicted in Figure 1D.(D) Normalized intensities of peptides corresponding to GPR133 and PTK7 as detected in the mass spectrometry analysis in Figure 1E, detailing all biological replicates.(E) Comparison of the GPR133-binding interaction of PTK7 vs. the previously reported ligand PLXDC2 using the Strep-Tactin purification paradigm. PTK7 and PLXDC2 (prey) were tagged with the Myc epitope for detection, while GPR133-H543R (bait) was tagged at the C terminus with TwinStrep tag for purification. Note that PTK7 co-purifies with GPR133, while PLXDC2 does not co-purify at detectable amounts under the same experimental conditions. See also Figure S2.