Q9 mini 18.02 sn software

To evaluate gene expression of arf3a and arf3b paralogs throughout zebrafish embryogenesis, total RNA was isolated from whole embryonic tissue samples at different stages of development (1–18 hpf) using TRIzol reagent (Invitrogen, 15596026). The first-strand cDNA was synthesized from total RNA using the SuperScript™ IV First-Strand Synthesis System (Thermo-Fisher, 18091050) according to the manufacturer’s protocol and DNAse treatment was performed to avoid genomic DNA contamination. RT-PCR was performed with specific primer sets annealing on conserved regions of arf3a and arf3b zebrafish paralogs (ENSDART00000103639.5 and ENSDART00000053775.3, respectively) using GoTaq® G2 Green Master Mix (Promega, M7822) (Supplementary Table 11). Amplification of the elongation factor 1α (eif1α) was used as a housekeeping control gene. Alliance Mini HD9 with Q9 Mini 18.02-SN software (Uvitec) was used for signal detection. Uncropped gels are provided in the Supplementary information.

COS-1 cells were seeded at 3 × 10⁵ in six-well plates and the following day was transfected with WT or mutant myc-tagged ARF3 expression constructs for 24 hours. A subset of transfected cells was then treated with CHX (10 μg/ml) (Sigma-Aldrich, C7698) for 3 and 6 h and with proteasome inhibitor MG132 (100 μM) (Sigma-Aldrich, C2211) or with the autophagy inhibitor bafilomycin A1 (200 nM) (Sigma-Aldrich, B1793) for 6 h to assess protein stability and degradation pathways. Alliance Mini HD9 with Q9 Mini 18.02-SN software (Uvitec) was used for chemiluminescence detection. Uncropped blots are provided in the Source data file and Supplementary information.