An ER-trapped GalNac-T activity reporter demonstrates that GalNac-Ts are active in the ER upon Src-induced redistribution. (A) Schematic of Muc-PTS GalNac-T activity reporter. (B and C) Immunofluorescence studies of Muc-PTS cotransfected in HeLa cells with inactive Src (SrcK-mCherry, containing K295M mutation) and staining with ER (PDI) (B) or Golgi (Giantin) (C) markers. (D and E) Immunofluorescence studies of Muc-PTS cotransfected in HeLa cells with active Src (SrcE-mCherry, containing E378G mutation) and staining with ER (D) or Golgi (E) markers. (F) SDS-PAGE analysis of Muc-PTS expressed alone in HEK293T cells or cotransfected with active (ER-GALNT2) or inactive (ER-GALNT2(H226D)) ER-targeted GalNac-T2. Muc-PTS was either immunoprecipitated with an anti-FLAG antibody or pulled down using HPL-conjugated agarose. (G) SDS-PAGE analysis of Muc-PTS pulled down using HPL-conjugated agarose after expression in HEK293T cells and treatment with EGF (100 ng/ml) for indicated times. (H) SDS-PAGE analysis of Muc-PTS coexpressed in HEK293T cells with either inactive Src (SrcK) or active SrcE (SrcE). Muc-PTS was either immunoprecipitated with an anti-FLAG antibody or pulled down using HPL-conjugated agarose. In all images nuclei were stained using Hoechst and colored blue. Bar, 10 µm.