Figure 3.

Encoding Non-Hydrolyzable Phosphonate Analogue (2) of pSer in Genetically Engineered Mammalian Cells

(A) Phosphonate analogue of phosphoserine (2) used in this study.

(B) Protein expression in the PSAT-KO. Coomassie-stained SDS-PAGE gel and western blot of purified GFP from HEK293/PSAT-KO overexpressing PSPH. Amino acid 2 was used at 10 mM.

(C) Separation on phos-tag SDS-PAGE gel followed by immunoblotting is consistent with incorporation of 2 in the HEK293/PSAT-KO cell line overexpressing PSPH. GFP, GFP(150pSer) and GFP(150[2]) standards were produced in E. coli as described previously (Rogerson et al., 2015).

(D) Quantification of the relative incorporation of 2 as a result of increasing concentration of 2 added to the cells. Data represent mean ± SEM for three biological replicates.

(E) Incorporation of 2 into GFP(150TAG) reporter at the genetically encoded site was verified by ESI-MS/MS.

See also Figure S3.