Full-length ARF1 was amplified and cloned into the prokaryotic expression plasmid pET-28b (Novagen, Madison, WI). The siRNA against ARF1, the transient and stable ARF1-overexpressing plasmids, as well as plasmids expressing the shRNA and sgRNA against ARF1, were obtained from TransheepBio (Shanghai, China). The inducible ARF1-knockdown plasmid, IQGAP1-overexpressing plasmid, and the plasmid expressing sgRNA against IQGAP1 were purchased from IGEbio (Guangzhou, China). Using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China), the plasmids pcDNA3.1-IQGAP1-∆RGCT-flag, pcDNA3.1-IQGAP1-∆RGCT/GRD-flag, and pcDNA3.1-IQGAP1-∆RGCT/GRD/IQ-flag were generated by PCR amplification from pcDNA3.1-IQGAP1 and cloned into the pcDNA3.1 vector. The mutant constructs for pcDNA3.1-ARF1T48S, pcDNA3.1-ARF1C159G, and pET28b-ARF1T48S were created by the Fast Mutagenesis System (TransGen Biotech, Beijing, China). Transfection and establishment of stable cell lines were carried out as described previously 21. The sequences of siRNA and primers for cloning and mutation are listed in Table S1.

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