All siRNA sequences as well as ON-TARGETplus Non-targeting Control Pool (D-001810-10) sequences were purchased from Dharmacon. Dynamin-2 or BiP (GRP78) or Rab5 and 7 were depleted using the following siRNA sequences: dynamin-2 antisense sequence, 5′-GACAUGAUCCUGCAGUUCAUU-3′; BiP siRNA, 5′-…CGAGUGACAGCUGAAGACAAGGGUA-3′; Rab5 siRNA, 5′-GGAAGAGGAGUAGACCUUA-3′; and Rab7 siRNA, 5′-GCUGCGUUCUGGUAUUUGA-3′. Cells were transfected with indicated siRNAs using either a Santa Cruz Biotechnology transfection reagent or an Amaxa Nucleofector (Lonza) electroporation system as previously described (Chavez et al., 2015 (link); Yazbeck et al., 2017 (link)).
ECs were cotransfected with WT-S1PR1-GFP, Y143D-S1PR1-GFP, or Y143F-S1PR1-GFP cDNAs along with GTPase-defective dynamin (K44A) or control vector using Amaxa reagent. 24 h after transfection, cells were serum starved and then processed either for imaging or for biochemical analysis (Chavez et al., 2015 (link)). The transfection efficiency for GFP was 90%, while efficiency for S1PR1, dynamin, and other cDNAs, which include BiP, ER-mCherry, HA-tagged S1PR1 cDNAs and S1PR1-Dendra2, ranged between 40% and 60%.
ECs were cotransfected with WT-S1PR1-GFP, Y143D-S1PR1-GFP, or Y143F-S1PR1-GFP cDNAs along with GTPase-defective dynamin (K44A) or control vector using Amaxa reagent. 24 h after transfection, cells were serum starved and then processed either for imaging or for biochemical analysis (Chavez et al., 2015 (link)). The transfection efficiency for GFP was 90%, while efficiency for S1PR1, dynamin, and other cDNAs, which include BiP, ER-mCherry, HA-tagged S1PR1 cDNAs and S1PR1-Dendra2, ranged between 40% and 60%.