Supplementary Figure 5: Cdc42 is deactivated locally by RICH1 and SH3BP1.

a, Confocal microscopy sections showing the transient increase in endogenous CIP4 and Endophilin on FEME carriers after 2 but not 10min incubation with 10μM ML141 (‘Cdc42i 1’). Images are representative of 6 captures from three independent biological experiments. b, Confocal microscopy section showing the decrease or not in endogenous Endophilin recruitment at the leading edge of resting cells overexpressing wild-type EGFP-tagged RICH2, SH3BP1 or OPHN1 but not their R291A, R312A and R409A respective GAP mutants (GAP*). Arrowheads point to Endophilin spots at the cell surface. Images are representative of 6 captures from three independent biological experiments. c,d, Recruitment of endogenous RICH1, Lpd and CIP4 in resting BSC1 cells depleted or not for Lamellipodin (Lpd KD), RICH1 and SH3BP1 (R+S DKD) or FBP17 and CIP4 (F+C DKD). Images are representative of 10 captures from three independent biological experiments. e, Pull-down using GST or GST-SH3 domains of Endophilin A2 or FBP17 and cell extracts expressing RICH1, RICH2, SH3BP1 or OPHN1 tagged with EGFP at their C-termini. Inputs correspond to 1 to 5% of the cell extracts. All experiments were repeated independently at least three times with similar results. Unprocessed original scans are provided in Supplementary Fig. 6. Bars, 5 μm.