Figure 4.
GlcNAcylated Milton coprecipitates with FHL2. (A) TRAK1-interacting proteins identified by SILAC-MS after IP of endogenous TRAK1 from HEK293T cells expressing either OGT or OGA. The OGT-expressing cells were also treated with 100 µM PUGNAc (an inhibitor of OGA) to maximize O-GlcNAcylation. Protein enrichment is plotted against peptide counts. Commonly detected contaminants were removed from the plot. (See Table S1 for the full list of proteins.) The constituents of the motor–adaptor complex and key hits (enriched in immunoprecipitates from cells expressing OGT) are labeled. FHL2 is marked by an arrow. (B and C) Endogenous TRAK1 was immunoprecipitated from HEK293T cells expressing OGT, OGA, or GFP (control). (B) Representative Western blots demonstrating the expression and co-IP efficiency of FHL2 with TRAK1 are shown for OGT-, OGA-, and GFP-expressing cells. (C) Quantification of co-IP efficiency of FHL2 with TRAK1 as the ratio of the intensity of the FHL2 band to that of TRAK1, normalized to the GFP condition. Immunoreactivity for endogenous FHL2 was enriched by ∼60% in the immunoprecipitates from cells expressing OGT. n = 3 independent transfections per condition. (D and E) OGT expression enhanced interaction of Myc-TRAK1 and FHL2-Flag coexpressed in HEK293T cells. HEK293T cells were cotransfected with Myc-TRAK1, FHL2-Flag, and OGT or OGA. After IP of FHL2-Flag, immunoblots (D) were probed for Myc-TRAK1, FHL2-Flag, OGT, HA-tagged OGA, and O-GlcNAc moieties (using the antibody RL2). Cells expressing GFP instead of FHL2-Flag were used as a control for nonspecific binding of Myc-TRAK1 to the Sepharose beads. The IP (E) was quantified as the ratio of the intensity of the Myc-TRAK1 band to that of the FHL2-Flag, normalized to the OGA condition. Myc-TRAK1 coprecipitated with FHL2-Flag strongly in the presence of OGT but weakly in the presence of OGA. n = 3 independent transfections per condition. (F) Interaction of Myc-TRAK1 and FHL2-Flag is independent of the actin cytoskeleton. Co-IP as in D, but cells were treated with DMSO or 0.05 µM LatA for 90 min before lysis. During IP, DMSO or 5 µM LatA was added to the IP buffer to prevent actin repolymerization in the lysate. Disruption of the actin cytoskeleton via LatA did not prevent the OGT-enhanced association of FHL2-Flag with Myc-TRAK1. (G and H) Interaction of Myc-TRAK1 and FHL2-Flag is dependent on the O-GlcNAcylation state of Myc-TRAK1. FHL2-Flag and OGT were coexpressed in HEK293T cells with either Myc-TRAK1WT or Myc-TRAK1QMut. (G) Representative Western blot of the FHL2-Flag immunoprecipitates. (H) Quantification of co-IP efficiency of Myc- TRAK1WT or Myc-TRAK1QMut with FHL2-Flag. Immunoprecipitates were quantified by measuring the ratio of the intensity of the Myc-TRAK1 band to that of the FHL2-Flag, normalized to the Myc-TRAK1WT condition. Although Myc-TRAK1WT coprecipitated with FHL2-Flag strongly in the presence of OGT, Myc-TRAK1QMut showed a 60% decrease in the FHL2-Flag interaction even in the presence of OGT. n = 6 from 3 independent transfections per condition. For all quantifications, P values are from ratio paired t tests. All data points are shown. Bars indicate mean ± SEM. For panels showing Western blots, molecular weights (in kD) are indicated on the right.

GlcNAcylated Milton coprecipitates with FHL2. (A) TRAK1-interacting proteins identified by SILAC-MS after IP of endogenous TRAK1 from HEK293T cells expressing either OGT or OGA. The OGT-expressing cells were also treated with 100 µM PUGNAc (an inhibitor of OGA) to maximize O-GlcNAcylation. Protein enrichment is plotted against peptide counts. Commonly detected contaminants were removed from the plot. (See Table S1 for the full list of proteins.) The constituents of the motor–adaptor complex and key hits (enriched in immunoprecipitates from cells expressing OGT) are labeled. FHL2 is marked by an arrow. (B and C) Endogenous TRAK1 was immunoprecipitated from HEK293T cells expressing OGT, OGA, or GFP (control). (B) Representative Western blots demonstrating the expression and co-IP efficiency of FHL2 with TRAK1 are shown for OGT-, OGA-, and GFP-expressing cells. (C) Quantification of co-IP efficiency of FHL2 with TRAK1 as the ratio of the intensity of the FHL2 band to that of TRAK1, normalized to the GFP condition. Immunoreactivity for endogenous FHL2 was enriched by ∼60% in the immunoprecipitates from cells expressing OGT. n = 3 independent transfections per condition. (D and E) OGT expression enhanced interaction of Myc-TRAK1 and FHL2-Flag coexpressed in HEK293T cells. HEK293T cells were cotransfected with Myc-TRAK1, FHL2-Flag, and OGT or OGA. After IP of FHL2-Flag, immunoblots (D) were probed for Myc-TRAK1, FHL2-Flag, OGT, HA-tagged OGA, and O-GlcNAc moieties (using the antibody RL2). Cells expressing GFP instead of FHL2-Flag were used as a control for nonspecific binding of Myc-TRAK1 to the Sepharose beads. The IP (E) was quantified as the ratio of the intensity of the Myc-TRAK1 band to that of the FHL2-Flag, normalized to the OGA condition. Myc-TRAK1 coprecipitated with FHL2-Flag strongly in the presence of OGT but weakly in the presence of OGA. n = 3 independent transfections per condition. (F) Interaction of Myc-TRAK1 and FHL2-Flag is independent of the actin cytoskeleton. Co-IP as in D, but cells were treated with DMSO or 0.05 µM LatA for 90 min before lysis. During IP, DMSO or 5 µM LatA was added to the IP buffer to prevent actin repolymerization in the lysate. Disruption of the actin cytoskeleton via LatA did not prevent the OGT-enhanced association of FHL2-Flag with Myc-TRAK1. (G and H) Interaction of Myc-TRAK1 and FHL2-Flag is dependent on the O-GlcNAcylation state of Myc-TRAK1. FHL2-Flag and OGT were coexpressed in HEK293T cells with either Myc-TRAK1WT or Myc-TRAK1QMut. (G) Representative Western blot of the FHL2-Flag immunoprecipitates. (H) Quantification of co-IP efficiency of Myc- TRAK1WT or Myc-TRAK1QMut with FHL2-Flag. Immunoprecipitates were quantified by measuring the ratio of the intensity of the Myc-TRAK1 band to that of the FHL2-Flag, normalized to the Myc-TRAK1WT condition. Although Myc-TRAK1WT coprecipitated with FHL2-Flag strongly in the presence of OGT, Myc-TRAK1QMut showed a 60% decrease in the FHL2-Flag interaction even in the presence of OGT. n = 6 from 3 independent transfections per condition. For all quantifications, P values are from ratio paired t tests. All data points are shown. Bars indicate mean ± SEM. For panels showing Western blots, molecular weights (in kD) are indicated on the right.

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