Sc 11049

To test the physical interactions between SCRIB and ZDHHC7, Flag-SCRIB and HA-ZDHHC7 were co-transfected into HEK293A cells. ZDHHC7-interacting proteins were immunoprecipitated by anti-HA magnetic beads (88836, Thermal Scientific), and SCRIB was detected in the complex by western blot using anti-SCRIB antibody (sc-11049, Santa Cruz, 1:1000). Similarly, SCRIB-interacting proteins were immunoprecipitated by anti-Flag magnetic beads (M8823, Sigma-Aldrich), and ZDHHC7 was detected in the complex by western blot using anti-HA antibody (H3663, Sigma Aldrich, 1:1000). To validate the interactions between endogenous SCRIB and ZDHHC7, HEK293A cells was transfected with HA-ZDHHC7 construct or vector control, and the cell lysate was subjected to immunoprecipitation using anti-HA magnetic beads. Endogenous SCRIB was detected in the IP samples by western blot using anti-SCRIB antibody (sc-11049, Santa Cruz, 1:1000). To determine the domain requirements for SCRIB to interact with ZDHHC7, Flag-SCRIB full-length or truncation mutants and HA- ZDHHC7 were co-transfected into HEK293A cells. ZDHHC7-interacting proteins were immunoprecipitated by anti-HA magnetic beads (88836, Thermal Scientific), and anti-Flag antibody (F1804, Sigma Aldrich, 1:2000) was used to detect Flag-SCRIB truncations.

Cells were lysed with RIPA buffer supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Roche). Lysates were denatured by heating for 5 minutes at 95 °C and loaded onto 4–12% Bis-Tris polyacrylamide gel. NuPAGE MOPS or MES running buffer (Invitrogen) was used for the SDS-PAGE. The proteins were subsequently transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked and incubated with primary antibodies and secondary HRP-conjugated antibodies, and developed by exposure to film. Antibody and dilutions used in the studies: anti-FLAG M2 (F1804, Sigma Aldrich, 1:5000), anti-HA (H3663, Sigma Aldrich, 1:1000), anti-GFP (sc-9996, Santa Cruz, 1:2000), anti-YAP (AT4556a, Abgent, 1:1000), anti-pYAP (13008S, Cell signaling, 1:1000), anti-SCRIB (sc-11049, Santa Cruz, 1:3000), anti-ZDHHC7 (ab138210, Abcam, 1:500), anti-AKT (4691S, Cell signaling, 1:1000), anti-pAKT(4060S, Cell signaling, 1:1000), anti-MEK(4694S, Cell signaling, 1:1000), anti-pMEK (9154S, Cell signaling, 1:1000), anti-H-Ras (sc-520, Santa Cruz, 1:1000), anti-GAPDH (2118S, Cell signaling, 1:1000), anti-β-actin (ab6276, Abcam, 1:500). Full images of blots are shown in the supplementary information (Supplementary Figs. 33–49)