Smurf1 has no effect on Talin-H degradation. (A) CFP-Talin-H plasmid (2 µg) was transfected into HEK293T cells together with increasing amounts of Smurf1 expression vector. Talin-H expression was determined by immunoblotting with an anti-GFP antibody 24 h after transfection. (B) Flag-Smurf1 was transfected into HEK293T cells together with Talin-H or mutants of Talin-H, and Talin-H expression was examined. (C) HEK293T cells transfected with Flag-Talin-H were treated with proteasome inhibitor MG132 (20 µM) or DMSO for 6 h, and Smurf1 and Flag-Talin-H protein expression were detected. (D) Flag-Talin-H and HA-Ub plasmids were cotransfected into HEK293T cells together with control vector, Myc-Smurf1 (WT), or Myc-Smurf1 (C699A) expression plasmid. Talin-H ubiquitination was detected by immunoprecipitation with anti-Flag M2 beads and immunoblotting with anti-HA antibody. (E) HEK293T cells were transfected with Flag-Talin-H, Myc-Smurf1, and K48R or K63R Ub mutant plasmids, and after 24 h, an in vivo ubiquitination assay was performed. (F) HEK293T cells were transfected with Flag-Talin-H, Myc-Smurf1, and different linkage Ub plasmids, and after 24 h, an in vivo ubiquitination assay was performed. (G) HEK293T cells were transfected with HA-Ub, Myc-Smurf1, and Flag-Talin-H K83+357 mutant plasmid, and then an in vivo ubiquitination assay was performed.
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