An α-cat ΔM1 mutant shows greatly enhanced association with LZTS2 than the α-cat R551A salt-bridge disrupting mutant. (A) Immunoblot/SDS-PAGE analysis of GFP-α-cat affinity pull-down assay to assess α-cat mutant association with LZTS2. Afadin is interrogated as a positive control based on data in Table S1. (B) Graph shows quantification of α-cat effector band intensity (i.e., LZTS2 or Afadin) normalized to GFP-α-cat mutant used for affinity precipitation. Note that loss of the α-cat M1-domain leads to enhanced association between α-cat and LZTS2, suggesting LZTS2 is a conformation-sensitive proximity partner of α-cat. Significance by one-way ANOVA where **** reflects P < 0.0001 for the α-cat ΔM1 mutant relative to all other mutants. The α-cat R551A mutant shows significantly greater association with LZTS2 than GFP-α-cat WT by unpaired t test (**P = 0.0026); R551A shows significantly greater association with Afadin than GFP-α-cat WT by unpaired t test (*P = 0.0270). Source data are available for this figure: SourceData FS7.