Fig. 5: C1GALT1 promotes O-glycosylation and stabilization of SMO in ES cells. | Nature Communications

Fig. 5: C1GALT1 promotes O-glycosylation and stabilization of SMO in ES cells.

From: The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma

Fig. 5

a PNA lectin pull-down assay. (Left) Schematic showing a PNA lectin bead (gray) bound to T antigen, comprising GalNAc (yellow square) and Gal (yellow circle), which is attached to SMO through serine or threonine (S/T). The O-glycosidase (O-glyc) cleavage site is shown. (Right) Representative immunoblot showing the level of O-glycosylated SMO, detected by either PNA lectin pull-down (PD) followed by SMO immunoblot (IB) analysis or SMO immunoprecipitation (IP) followed by PNA IB, in A673 cell lysates treated with O-glycosidase and α2-3,6,8 neuraminidase, which removes terminal sialic residues that inhibit lectin binding and O-glycosidase cleavage. b Representative co-IP assay in A673 cells monitoring the presence of SMO in a C1GALT1 IP, performed using a C1GALT1 antibody (ab), or vice versa. c Representative immunoblot showing levels of O-glycosylated SMO, total SMO, GLI1, EWSR1::FLI1 (monitored using an anti-FLI1 antibody), and C1GALT1 in A673 cells treated with 100 nM ITZ. d (Top) Representative immunoblot showing SMO levels in a cycloheximide (CHX) chase assay performed in A673 cells treated with DMSO or 100 nM ITZ. (Bottom) Quantification; time 0 in the presence of DMSO or ITZ was set to 100%. The plot shows the average SMO levels from two independent experiments. e Representative immunoblot showing SMO levels in A673 cells stably expressing an NS or C1GALT1 shRNA (left) or treated with DMSO or 100 nM ITZ for 72 h (right) and treated in the presence or absence of MG132 or bafilomycin A1 (BafA1). f Representative immunoblot showing the level of O-glycosylated SMO in A673 cells stably expressing an NS or C1GALT1 shRNA and treated in the presence or absence of MG132 or BafA1. g Representative immunoblot showing FLAG-SMO levels in A673 cells stably expressing FLAG-tagged wild-type SMO, SMO(T55A) or SMO(T500A) in the presence or absence of MG132 or BafA1. h Representative ubiquitination assay showing the levels of polyubiquitinated SMO(T55A) or SMO(T500A) in A673 cells expressing HA-tagged ubiquitin (HA-Ub) and treated in the presence or absence of MG132 or BafA1. Source data are provided as a Source Data file.

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