Visualizing Cell Surface TGF-β Receptors

Cell surface TGF-β receptors were visualized by cell surface protein biotinylation as described [49 (link)]. Briefly, NMuMG cells were grown to confluence, serum starved for 4–6 h, treated with 2 ng/ml TGF-β for 30 min to 1 h, and labeled with Sulpho-NHS-LC Biotin (Thermo Scientific) at 4°C for 30 min. Cells were washed with 100 mM glycine and lysed in lysis buffer. Biotinylated cell surface proteins were adsorbed to neutravidin agarose (Thermo Scientific) and analyzed by immunoblotting with antibodies to TβRI or TβRII.

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Muthusamy B.P., Budi E.H., Katsuno Y., Lee M.K., Smith S.M., Mirza A.M., Akhurst R.J, & Derynck R. (2015). ShcA Protects against Epithelial–Mesenchymal Transition through Compartmentalized Inhibition of TGF-β-Induced Smad Activation. PLoS Biology, 13(12), e1002325.

Publication 2015

Sulpho-NHS-LC Biotin neutravidin agarose

Agarose Antibodies Biotinylation Buffer Cell surface protein Cell surface proteins Cell surface receptors Cells Glycine Neutravidin Serum Sulpho nhs lc biotin Tgf β Tgf β 2 Tβrii

Corresponding Organization : UCSF Helen Diller Family Comprehensive Cancer Center

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Variable analysis

independent variables

Treatment with 2 ng/ml TGF-β for 30 min to 1 h

dependent variables

Cell surface TGF-β receptors (TβRI and TβRII) visualized by cell surface protein biotinylation

control variables

NMuMG cells grown to confluence
NMuMG cells serum starved for 4–6 h
Labeling with Sulpho-NHS-LC Biotin at 4°C for 30 min
Washing with 100 mM glycine
Lysis in lysis buffer
Biotinylated cell surface proteins adsorbed to neutravidin agarose

controls

No positive or negative controls explicitly mentioned

Annotations

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