Mcherry clathrin lc 15

mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson (Rizzo et al., 2009 (link)); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama (Makyio et al., 2012 (link)); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius (Hayer et al., 2010 (link)); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla (Várnai and Balla, 1998 (link)).

Plasmids for tracking PI species were from Addgene. Specifically, mCherry-Clathrin LC-15 (Addgene #55019) [71 (link)]; PLCD1(PH)-mCherry (Addgene #36075) [72 (link)]; PH-PLCD1-GFP (Addgene #51407), PH-PLCD1(R40L)-GFP (Addgene #51408) [73 (link)]; pLentiLifeACT-EGFP BlastR (Addgene #84383) [74 (link)]; PH-Btk-GFP (Addgene #51463), GFP-PH-TAPP1 (Addgene #161985); and pLenti-EGFP-P4M-SidMx2 (Addgene #136997), pLenti-EGFP-2xFYVE (Addgene #136996) were gifts from Ken-Ichi Takemaru. Plasmids were also constructed by Vectorbuilder: VB200917 is a lenti-mCherry-shCLTC containing U6-driven shRNA sequence 5′-CGTGTTCTTGTAACCTTTATT-3′. VB200917 is a lenti-mCherry-shLuciferase containing U6-driven shRNA sequence 5′- ATGTTTACTACACTCGGATAT-3′ and was used to create lentivirus to infect MCF7, 4226, and MPE600 cell lines. Infected cells were sorted for mCherry using FACS as above and tested for engulfment. Human PIK3C2B-GFP fusion overexpression vector is a lentiviral CMV-driven plasmid from VectorBuilder. miRFP-713 (near-infrared fluorescent protein) overexpression vector is a lentiviral EFS-driven plasmid from VectorBuilder. 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder. Lentiviral EFS-driven LYN11 mCherry fusion protein construct was also produced by VectorBuilder.

EGFP-Rab5 (Addgene) was a gift from Arnab Gupta. Cells were transfected with 1 μg of Rab5 plasmid DNA to label early endosomes, respectively, by using Lipofectamine 3000 (Invitrogen). EGFP-CAAX [41 (link)] was a gift from Lei Lu. Cells were transfected with 1 μg EGFP-CAAX (Addgene) plasmid to mark the membrane. Cells were transfected with 1 μg of mCherry- Clathrin LC-15 (Addgene) to label clathrin-coated vesicles. To mark fast recycling endosomes, cells were transfected with 1 μg of mCherry- Rab 4a plasmid. Other treatments, if required, were performed 16 h after transfection.
Fixation of cells was performed by using 4% paraformaldehyde (Sigma-Aldrich) for 15 min at 37°C temperature.