Fig. S4.
Binding and ubiquitination activities of FBXO27-related F-box proteins to LAMP1 and LAMP2. (A) Glycoprotein-specific F-box proteins bind to LAMP1 and LAMP2. 293T cells were transfected with plasmids encoding either only FLAG-tag alone, or one of the following C-terminally FLAG-tagged proteins, FBXO27, FBXO27G2M, FBXO27FW/AA, FBXO2, FBXO6, FBXO17, or FBXO44, in combination with SKP1, and cells were treated with or without LLOMe for 2 h before harvesting. WCLs were subjected to immunoprecipitation with an anti-FLAG antibody, and analyzed by immunoblotting with the indicated antibodies. (B) Ubiquitination assay for LAMP1 and LAMP2. 293T cells were transfected with plasmids encoding FLAG–TR-TUBE and either empty vector control, FBXO27, FBXO27G2M, FBXO27FW/AA, FBXO2, FBXO6, FBXO17, or FBXO44, and subsequently treated with LLOMe for 2 h before harvesting. Cell lysates were immunoprecipitated with anti-FLAG antibody, and analyzed by immunoblotting. Vertical bars and arrows denote the positions of ubiquitinated and unmodified LAMPs, respectively.