Figure S3.
Lpg2525 preferentially ubiquitinates GTP-bound active Rab5. (a) HeLa-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Rab5A (S34N) or (Q79L), HA-UB, and GFP or GFP-Lpg2525. At 24 h after transfection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against HA, GFP, and FLAG. (b) HeLa-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Rab5A (S34N) or (Q79L) and GFP-Lpg2525. At 24 h after transfection, cells were fixed and stained with an antibody against FLAG. Bar, 5 µm. (c) HeLa-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Rab5A (Q79L) and GFP-Lpg2525. At 24 h after transfection, cells were fixed and stained with antibodies against UB and FLAG. Bars, 5 µm. An enlarged image of the white inset box is shown. (d) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting Cullin1 or SKP1. At 72 h after transfection, the efficacy of siRNAs was assessed by IB with Cullin1 and SKP1 antibodies. IB with GAPDH antibody was conducted as an internal control. (e) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting Cullin1 or SKP1. At 48 h after transfection, the cells were transfected with an additional plasmid-encoding 3x-FLAG-Rab5A (Q79L), GFP-Lpg2525, and HA-UB for 24 h, lysed, and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with the indicated antibodies. IB, immunoblotting. Source data are available for this figure: SourceData FS3. Refer to the image caption for details.

Lpg2525 preferentially ubiquitinates GTP-bound active Rab5. (a) HeLa-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Rab5A (S34N) or (Q79L), HA-UB, and GFP or GFP-Lpg2525. At 24 h after transfection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against HA, GFP, and FLAG. (b) HeLa-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Rab5A (S34N) or (Q79L) and GFP-Lpg2525. At 24 h after transfection, cells were fixed and stained with an antibody against FLAG. Bar, 5 µm. (c) HeLa-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Rab5A (Q79L) and GFP-Lpg2525. At 24 h after transfection, cells were fixed and stained with antibodies against UB and FLAG. Bars, 5 µm. An enlarged image of the white inset box is shown. (d) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting Cullin1 or SKP1. At 72 h after transfection, the efficacy of siRNAs was assessed by IB with Cullin1 and SKP1 antibodies. IB with GAPDH antibody was conducted as an internal control. (e) HeLa-FcγRII cells were transfected without (mock) or with siRNA-targeting Cullin1 or SKP1. At 48 h after transfection, the cells were transfected with an additional plasmid-encoding 3x-FLAG-Rab5A (Q79L), GFP-Lpg2525, and HA-UB for 24 h, lysed, and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with the indicated antibodies. IB, immunoblotting. Source data are available for this figure: SourceData FS3.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal