Kinase Assay and Immunoprecipitation Protocol

In vitro kinase assay, mammalian cell culture, transfection, immunoprecipitation, 32P orthophosphate metabolic labeling, as well as the antibodies used in this study were described previously (Cui et al., 2015 (link)). U2OS cells (RRID: CVCL_0042) were originally obtained from and authenticated by the American Type Culture Collection (ATCC, Manassas, VA). Rabbit anti-V5 polyclonal antibody (Millipore, Billerica, MA; AB3792; RRID: AB_91591) was used for immunoprecipitation. Monoclonal anti-V5 antibody (Life Technologies, Carlsbad, CA; R960-25; RRID: AB_2556564) and anti-FLAG antibody (Sigma, St. Louis, MO; F3165; RRID: AB_259529) were used for Western blotting.

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Cui J., Zhu Q., Zhang H., Cianfrocco M.A., Leschziner A.E., Dixon J.E, & Xiao J. (2017). Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding. eLife, 6, e23990.

Publication 2017

Monoclonal anti-V5 antibody anti-FLAG antibody

Anti antibody Antibodies Assay Cell culture Cells Immunoprecipitation Kinase Mammalian Monoclonal antibody Orthophosphate Rabbit Transfection

Corresponding Organization :

Other organizations : University of California, San Diego, Center for Life Sciences, Peking University

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Variable analysis

independent variables

Transfection

dependent variables

Kinase activity
Metabolic labeling

control variables

Cell line (U2OS cells)
Antibodies used in this study

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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