Fig. 5: O-GlcNAcylation of AMFR impairs AMFR-TSPAN4 interactions.
From: O-GlcNAcylation of AMFR stabilizes TSPAN4 to regulate migrasome formation for viral release
a HEK293T cells were transfected with AMFR-Flag or TSPAN4-Flag and OGT-Myc. Flag antibody immunoprecipitated or cell lysates were subjected to WB (n = 3 independent experiments). O-GlcNAc antibody RL2 was used. Source data are provided as a Source Data file. b Co-immunoprecipitation assay for the interactions between AMFR-HA and OGT-Flag in HEK293T cells (n = 3 independent experiments). Source data are provided as a Source Data file. c GST pull-down analysis of GST-OGT with His-AMFR (n = 3 independent experiments). Source data are provided as a Source Data file. d OGT directly O-GlcNAcylates AMFR in vitro. In vitro glycosylation assay was performed with His-AMFR as substrate, which was incubated with purified GST-OGT, and the GlcNAcylated AMFR was blotted with RL2 antibody and the total His-AMFR and purified GST-OGT proteins were detected by WB (n = 3 independent experiments). Source data are provided as a Source Data file. e AMFR O-GlcNAcylation is reversed by OGA via in vitro glycosylation assay (n = 3 independent experiments). Source data are provided as a Source Data file. f HEK293T cells were transfected with or without OGT siRNA for 12 h, and cells were further transfected with AMFR-Flag for 36 h. Cell lysates were subjected to immunoprecipitation with Flag antibody and analyzed via WB (n = 3 independent experiments). Source data are provided as a Source Data file. g HEK293T cells were transfected with plasmids as indicated. Cell lysates were subjected to immunoprecipitation with Flag antibody and analyzed via WB (n = 3 independent experiments). Source data are provided as a Source Data file. h Diagram showing the amino acid sequence of AMFR and six potential O-GlcNAcylated sites. i HEK293T cells were transfected with indicated plasmids, and AMFR O-GlcNAcylation was analyzed by immunoprecipitation with Flag antibody and WB with the indicated antibodies. Quantification signal of AMFR and its mutants O-GlcNAcylation of immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. j Purified WT or His-AMFRT643A was used as substrates for in vitro glycosylation assay (n = 3 independent experiments). Source data are provided as a Source Data file. k LC-MS/MS spectrum of the O-GlcNAcylated peptide of AMFR. The Y1 ions confirm that O-GlcNAcylation occurs at T643. l, m NRK-ITGA5-mCherry cells were transfected with indicated plasmid. Cells were imaged by confocal microscopy. Green, GFP and OGT-GFP; red, ITGA5-mCherry; white, Halo, AMFR-Halo and AMFRT643A-Halo. Number of migrasomes from the cells in (l); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. n HeLa cells stably expressing TSPAN4-Flag were transfected with indicated plasmids for 24 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. o HEK293T cells were transfected with or without OGT siRNA for 12 h, and cells were further transfected with AMFR-Myc and TSPAN4-Flag for 36 h. Cell lysates were subjected to immunoprecipitation with Flag antibody and analyzed via WB. Quantification signal of AMFR-Myc of co-immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. p HEK293T cells were transfected with the indicated plasmids for 36 h. Cell lysates were subjected to immunoprecipitation with Flag antibody and analyzed via WB. Quantification signal of AMFR-HA of co-immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. q HEK293T cells were transfected with the indicated plasmids for 36 h. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed via WB. Quantification signal of AMFR-Flag and its mutant of co-immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. r O-GlcNAcylation of AMFR decrease AMFR-TSPAN4 interactions in vitro. His-AMFR was first incubated with OGT and UDP-GlcNAc for 4 h, then incubated with GST-tagged TSPAN4. Immunoprecipitation was performed with anti-His antibody. Quantification signal of GST-TSPAN4 of Pull-down from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file.
