MCF7 cells (from the American Type Culture Collection, ATCC) were
grown in DMEM medium (Sigma-Aldrich) containing 10% fetal bovine serum
(FBS, Thermo) at 37 °C with 5.0% CO2 in a humidified
incubator. When the confluency reached 80%, the medium was replaced
using a heavy lysine (K8) and arginine (R6) containing medium, and
250 μM N-azidoacetylgalactosamine-tetraacetylated
(Ac4GalNAz, Click Chemistry Tools) and 50 μM puromycin
(Puro, Santa Cruz Biotechnology) were added to the medium. For the
control samples, Puro was not added. Then, cells were treated for
1 h. For the boosting sample, cells were cultured in the medium containing
heavy lysine and arginine for 2 weeks for complete labeling of proteins
with heavy K and R in the cells. Then the cells were treated with
250 μM Ac4GalNAz for 48 h to label O-GlcNAcylated proteins.
Cells from different samples were harvested
and washed with ice-cold PBS twice. They were lysed with a buffer
containing 50 mM HEPES, pH = 7.4, 150 mM NaCl, 0.5% SDC, 0.1% SDS,
1% NP-40, 50 μM Thiamet G, 50 units/mL Benzonase nuclease (Millipore),
and 1 tablet/10 mL EDTA-free protease inhibitor for 2 h at 4 °C.
Then the lysates were centrifuged for 10 min at 4696g, and the debris was discarded. The labeled glycopeptides or glycoproteins
were reacted with a biotin probe through a click chemistry reaction.
In the cell lysate, 250 μM photocleavable (PC) biotin-alkyne
(Click Chemistry Tools), 1 mM CuSO4, 5 mM Tris(3-hydroxypropyltriazolylmethyl)
amine (THPTA, Click Chemistry Tools), 5% dimethyl sulfoxide (DMSO),
15 mM sodiuml -ascorbate (Sigma), and 15 mM aminoguanidine
hydrochloride (Sigma) were added, and the reaction lasted for 2 h
at room temperature.
grown in DMEM medium (Sigma-Aldrich) containing 10% fetal bovine serum
(FBS, Thermo) at 37 °C with 5.0% CO2 in a humidified
incubator. When the confluency reached 80%, the medium was replaced
using a heavy lysine (K8) and arginine (R6) containing medium, and
250 μM N-azidoacetylgalactosamine-tetraacetylated
(Ac4GalNAz, Click Chemistry Tools) and 50 μM puromycin
(Puro, Santa Cruz Biotechnology) were added to the medium. For the
control samples, Puro was not added. Then, cells were treated for
1 h. For the boosting sample, cells were cultured in the medium containing
heavy lysine and arginine for 2 weeks for complete labeling of proteins
with heavy K and R in the cells. Then the cells were treated with
250 μM Ac4GalNAz for 48 h to label O-GlcNAcylated proteins.
Cells from different samples were harvested
and washed with ice-cold PBS twice. They were lysed with a buffer
containing 50 mM HEPES, pH = 7.4, 150 mM NaCl, 0.5% SDC, 0.1% SDS,
1% NP-40, 50 μM Thiamet G, 50 units/mL Benzonase nuclease (Millipore),
and 1 tablet/10 mL EDTA-free protease inhibitor for 2 h at 4 °C.
Then the lysates were centrifuged for 10 min at 4696g, and the debris was discarded. The labeled glycopeptides or glycoproteins
were reacted with a biotin probe through a click chemistry reaction.
In the cell lysate, 250 μM photocleavable (PC) biotin-alkyne
(Click Chemistry Tools), 1 mM CuSO4, 5 mM Tris(3-hydroxypropyltriazolylmethyl)
amine (THPTA, Click Chemistry Tools), 5% dimethyl sulfoxide (DMSO),
15 mM sodium
hydrochloride (Sigma) were added, and the reaction lasted for 2 h
at room temperature.
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