Eea1 tagrfp t

Wild-type HeLa cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198)^{6 (link)}; EEA1 TagRFP-T, a gift from Silvia Corvera (Addgene plasmid #42635)^{24 (link)}; EGFP-EEA1, a gift from Silvia Corvera (Addgene plasmid #42307)^{13 (link)}; EGFP-Rab5, a gift from Marci Scidmore (Addgene plasmid #49888); and mRFP-Rab5, a gift from Ari Helenius (Addgene plasmid #14437)^{69 (link)}. Cells were transfected with a total of 1 µg DNA (0.3 µg + 0.3 µg plasmid of interest + 0.4 µg blank DNA) using lipofectamine 3000 (Thermo Fisher Scientific). The DNA sequence for the N-terminal mutant of EEA1 carrying F41A and I42A, deficient in Rab5 binding, was synthesised and cloned into the TagRFP-T vector using the XhoI/BamHI sites. For the C-terminal binding mutant carrying R1375A, the synthesised sequence was cloned into the TagRFP-T vector using the XhoI/BamHI sites. It has been reported that drastic over-expression of APPL1 or EEA1 results in colocalisation of APPL1 and EEA1 on Rab5 endosomes; we, therefore, optimised this concentration by screening for this artefact and choosing conditions where we observed no overlap of APPL1 and EEA1.

Cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198)^{23 (link)}, EGFP-APPL1-ΔPTB, a gift from Donna Webb (Addgene plasmid #59768)^{39 (link)}, DsRed-p150 217-548, a gift from Trina Schroer (Addgene plasmid #51146)^{29 (link)}, EEA1 TagRFP-T, a gift from Silvia Corvera (Addgene plasmid #42635)^{50 (link)}, pEGFR-PATagRFP, a gift from Vladislav Verkhusha (Addgene plasmid #31950)^{51 (link)}. Cells were transfected with a total of 1 µg DNA (plasmid of interest—0.2 µg, blank DNA—0.8 µg) for single protein expression or (plasmids of interest—0.2 µg + 0.2 µg, blank DNA—0.6 µg) using lipofectamine 3000 (Thermo Fisher Scientific) or the Neon electroporator (Invitrogen) at 1200 mV 20 ms, 2 pulses for RPE1 and 1005 mV 35 ms, 2 pulses for HeLa. HeLa cells were ensured for mild expression by the following: The transfection mix consisted of APPL1 or EEA1 plasmids adding up to 20% of the total DNA amount for transfection, with the rest consisting of blank DNA. Secondly, it has been reported that overexpression of APPL1 or EEA1 results in colocalization of APPL1 and EEA1 on Rab5 endosomes. We optimized this concentration by screening for this artifact, where we see no overlap of APPL1 and EEA1. Thirdly, APPL1 overexpression impairs EGFR internalization. However, in all our experiments, EGF/EGFR complex is trafficked efficiently to the peri-nuclear region.