Abstract
This protocol describes experimental procedures for in vitro dephosphorylation assay of human protein c-Myc. This protocol can be adapted to detect phosphatase activity of other Ser/Thr phosphatases.
Keywords: Dephosphorylation, c-Myc, SCP1
Background
Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1 (SCP1, also known as CTDSP1 or NLI-IF) belongs to the FCP/SCP phosphatase family and was originally reported to dephosphorylate the C-terminal domain (CTD) of RNA polymerase II (Yeo et al., 2003). Smad2, 3 (Wrighton et al., 2006), Snail (Wu et al., 2009), PML (Lin et al., 2014), and c-Myc (Wang et al., 2016) have also been identified as substrates of SCP1.
Materials and Reagents
Equipment
Procedure
Data analysis
HA-c-Myc purified from HEK293T cells was incubated with purified GST, GST-SCP1-WT, and GST-SCP1-DN proteins at 37 °C for 30 min. The immunoprecipitates are analyzed using anti-pSer62, anti-c-Myc, or anti-GST antibodies. SCP1 dephosphorylates c-Myc at Ser62 in vitro, analyzed using Western blotting. Figure 1. SCP1 dephosphorylates c-Myc at Ser62 in vitro (Wang et al., 2016)
Recipes
Acknowledgments
This work was supported by grants from National Natural Science Foundation of China (31501141).
References
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