Supplemental Experimental Procedures

tained by PCR, sequenced, and cloned into pCMUIV vector [S1]. Dominant-negative Rab11A S25N and Rab4B S22N mutants were cloned similarly into pCMUIV vector with endogenous Kozak se-quences. Myc-tagged human Rab35 cDNA was expressed from pcDNA3 vector (Invitrogen). PLC-d-PH-GFP-expressing plasmid was described in [S2]. pCMUIV-Rab35 S22N plasmid mutated by silent mutation with QuickChange (Stratagene) to render this con-struct insensitive to siRNA. For this purpose, the Rab35 50-GCTCAC GAAGAACAGTAAA-30 sequence was mutated into the 50-GCTCAC GAAAAATTCTAAA-30 sequence. Rabbit antisera against recombinant full-length human Rab35 taggedwith GST and taggedwith 6Histidine weremade by Eurogen-tec and Agrobio, respectively, and affinity-purified on untagged recombinant full-length human Rab35 protein was prepared as described in [S3]