Reagents and Constructs

70 kDa TMR-Dextran and FITC-Dextran were purchased from Fina Biosolutions (Rockville, MD). All chemicals were molecular biology grade. Bafilomycin A1, Dynasore, 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA), LY294002, U0126, KH7, H89, and S0859 were purchased from Cayman Chemical Co (Ann Arbor, MI). Cholesterol-water soluble reagent was purchased from Sigma (cholesterol-methyl-beta-cyclodextran). Non-targeting siRNA pool #1 (D-001206–13), ATP6V1A 3’ UTR-targeting siRNA (Sense: 5’-GCA AUG GUU UGU UGA GAU AUU-3’), siV1A (MU-017590–00), siV0a3 (M-012198–01), siKRas (M-005069–00), siRac1 (M-003560–06), siSLC4A7 (M-007586–01), siV1B2 (M-011589–01), siV0a1 (M-017618–01), siV1E1 (M-011590–01), and siV0c (M-017620–02) were purchased from GE Dharmacon (Lafayette, CO). A complete list of siRNA sequences can be found in Supplementary Table 2.

pCGT was used as a mammalian expression vector to express T7-HRasV12 and T7-KRasV12. pTRIPZ lentiviral inducible shRNA plasmid was from GE Dharmacon. pEGFP-Rac1WT was generated as previously described²⁵. GFP-Rac1^L61, GFP-Rac1^L61 K-tail, and R-pre constructs were kindly provided by Dr. Mark Philips²⁶. The R-pre construct contains a modified sequence of the membrane targeting domain of KRas linked to red fluorescent protein.

V1A-FLAG construct was cloned from HeLa cDNA into pCMV-3tag-8 vector backbone using the following primers:

fw 5’ – GGAGGACTCGAGACCAGTATGGATTTTTCCAAGCTACCC – 3’

rv 5’ – ACCACCGGATCCTTCAAATCTTCAAGGCTACGGAATGC – 3’.

V1A-GFP11 construct was created by PCR amplifying GFP11 from pEGFP-GFP11-Clathrin light chain. pEGFP-GFP11-Clathrin light chain was a gift from Bo Huang (Addgene plasmid #70217). The following primers were used:

fw 5’ – TCATCAGCGGCCGCGTCGCCACCATGTCGGGAGGTT – 3’

rv 5’ – ATTAATGCGGCCGCCTATCCGGATCCGCCTGTAATCCCAGC – 3’.

The PCR amplified GFP11 was then cloned into the V1A-FLAG construct. The reverse primer introduced a stop codon prior to the c-terminal FLAG-tag. The first NotI site was destroyed and GFP11 was put into frame with V1A by site-directed mutagenesis using the following primers:

fw 5’ – CGTAGCCTTGAAGATGCCGGCCGCGTCGCCACC – 3

rv 5’ – GGTGGCGACGCGGCCGGCATCTTCAAGGCTACG – 3’.

Lyn-GFP1–10 construct was created by cloning a Lyn-tail sequence into pcDNA3.1-GFP(1–10). pcDNA3.1-GFP(1–10) was a gift from Bo Huang (Addgene plasmid # 70219). The following oligonucleotides were used:

fw 5’ – AGCTTGCCACCATGGGATGTATTAAATCAAAAAGGAAAGACGGGACAG – 3

rv 5’ – AATTCTGTCCCGTCTTTCCTTTTTGATTTAATACATCCCATGGTGGCA – 3’.

Lyn-tailed mCherry-SEpHluorin was a gift from Sergio Grinstein (Addgene plasmid #32002).

Constitutively-active PKA was created by mutating His87 to Gln in a GFP-tagged PKA catalytic subunit construct using site-directed mutagenesis according to the manufacturer’s instructions (Quickchange™, Agilent Technologies, Santa Clara, CA)²⁷.

pTRIPZ FLAG-KRasV12 was constructed by inserting a human codon optimized FLAG-tagged KRasV12 into the unique Age1, Mlu1 sites of pTRIPZ, which simultaneously removed RFP and the shRNA targeting region.