Reporting the phosphorylation status of a substrate protein using genetically encoded Uaas. a) Schematic illustration. The broken line indicates that the phosphorylated residue and the fluorescent Uaa can be on the same or separate proteins. b) Crystal structure of the STAT3β homodimer depicting two monomers (pink and cyan) binding DNA (grey) (PDB 1BG1). c) Region framed in (b) illustrating the location of pTyr705 (red) in relation to Trp564 (green). d) Structure of L-(7-hydroxycoumarin-4-yl) ethylglycine (7HC).

7HC-STAT3β, similar to wt STAT3β, can be phosphorylated and binds a consensus DNA sequence. a) Western blot analysis of E. coli lysates from cells expressing STAT3β(564TAG) and the 7HC-specific tRNA/synthetase pair. b) Photograph of SDS-PAGE analysis of wt STAT3β and 7HC-STAT3β(564TAG) expressed in the presence and absence of 7HC. The gel was exposed to 365 nm UV light. (c) Western blot of protein samples incubated with and without Src kinase. Probing with a STAT3-specific antibody ensured that comparable amounts of STAT3 were loaded. (d) EMSA using ³²P labelled hSIE DNA probe. Probe incubated with wt STAT3β and 7HC-STAT3β was upshifted indicating that both proteins bind the hSIE probe. Specific competition was seen with excess unlabeled probe yielding dissociation constants for wt STAT3β (K_d = 6.3 ± 0.6 nM, n = 4) and 7HC-STAT3β (K_d = 6.8 ± 1.6 nM, n = 4).

7HC-STAT3β reversibly reports the phosphorylation status of STAT3. a) Fluorescence emission of 7HC-STAT3β before and after phosphorylation by Src kinase followed by dephosphorylation by CIP. b) Fluorescence emission of 7HC-STAT3β mutants Y705F and R609Q before and after phosphorylation by Src kinase. c) Western blots for 7HC-STAT3β and mutants using an antibody against phosphorylated STAT3. Equivalent amounts of STAT3 protein were loaded in each lane.

7HC in the 7HC-STAT3β protein experiences a pH change upon phosphorylation. a) Fluorescence excitation spectra of 7HC in aqueous buffer with emission recorded at 450 nm. b) Fluorescence emission spectra of 7HC in aqueous buffer with excitation at 363 nm. c) Fluorescence excitation spectra of 7HC-STAT3β with emission recorded at 450 nm before and after phosphorylation by Src kinase.

7HC-STAT3β reports the phosphorylation status of endogenous STAT3 from HepG2 cells. a) Western blot showing that STAT3 was phosphorylated in the nucleus of HepG2 cells only when activated by IL-6. b) Fluorescence increase of 7HC-STAT3β upon incubation with nuclear extracts. The values (± s.e.m.) were: IL-6 (−) 1.4 ± 0.2 and IL-6 (+) 5.9 ± 0.8, n = 3 from 3 independent batches of cells. The IL-6 activated nuclear fraction was statistically different from the uninduced sample (Student’s t-test, two-tailed, unpaired). c) Fluorescence emission spectra and d) excitation spectra of 7HC-STAT3β after incubation with the nuclear extracts of HepG2 cells with (+) and without (−) IL-6 induction. e) Western blot showing that 7HC-STAT3β was not phosphorylated by cell lysates. 7HC-STAT3β mixed with cell lysates before (t = 0) and after (t = 2 hr) incubation were analyzed. 7HC-STAT3β was N-terminally truncated and thus ran at a different position from endogenous STAT3. The blot was also probed with the penta-His antibody to detect the C-terminal His6 tag appended on 7HC-STAT3β.