Fig 2: The PLCγ1 cSH2 domain is required for the binding of PLCγ1 to phosphorylated pK15 in vitro and may serve as a dominant-negative inhibitor.GST-fused pK15 cytoplasmic tail WT (A), or its truncated mutant N2 [51] (B), were phosphorylated in vitro by 2xStrep-Src and an AlphaLISA was performed with the indicated His-tagged fragments of PLCγ1. Mean and standard deviation from at least two independent experiments with two technical replicates each are depicted. GST fusion proteins of the N1 (C) or N2 (D) truncation mutants of the pK15 cytoplasmic tail [51] were phosphorylated in vitro by 2xStrep-Src and their interaction with recombinant purified PLCγ1 tSH2 (tandem SH2) or SA (γ-specific array) domains was measured by AlphaLISA in the presence of increasing concentrations (300 nM, 600 nM, 900 nM, 1200 nM) of the purified PLCγ1 cSH2. Mean and a standard deviation from at least three independent experiments with two technical replicates each are depicted.–: empty vector (pTriex4). (E) An expression vector (PFJ) expressing full length pK15 or empty PFJ (EV) were co-transfected in HEK-293T cells together with the isolated cSH2 domains of PLCγ1 or PLCγ2 or their empty vector pTriex4. After 48h lysates were collected and analysed by Western blot using an antibody to PLCγ1 pY783 to assess the level of PLCγ1 phosphorylation. (F) The intensities of the pPLCγ1 bands shown in panel E were quantified by densitometry, normalized to the values obtained with the empty vector (EV) in the presence of pK15, and plotted as relative values. The diagram shows the results obtained in three independent experiments.

Fig 3: Ascorbic Acid binds to TET2 and increases its activity in cell free conditions.a, b Surface plasmon resonance (SPR) detection of AA [0–1000 µM] binding to purified recombinant GST-TET2CD captured on CM5 biosensor chip immobilized with anti-GST antibody in the presence of a fixed concentration of 5mC-DNA substrate (b) or GST tag free TET2CD captured by 5mC-DNA-biotin immobilized on SA chip (b), in the presence of NOG (100 µM) and Fe3+ (100 µM). The response curve in the absence of AA was used as control and subtracted to generate final response curve. Kd = koff/kon. c. TET2CD was used in an ELISA based assay with varying concentration of AA to determine the dose response and EC50 = 13.8±1.3 µM was calculated using Log(agonist) vs. response (R2 = 0.94) option in GraphPad Prism 8.0.2. d–e. Cell-free DNA combined with purified TET2 (20 nm) and AA (25 µM) for 2 h in the presence of Fe2+ or Fe3+ (100 µm). d dot blot. Top right: 20 pmol 5hmC labeled DNA and its serial twofold dilution were used as positive control. e 2D-UPLC-MS/MS. Statistical significance (p values) from two tailed t test are indicated, ns not significant.

Fig 4: Contribution of individual PLCγ1 SH2 domains to the interaction with phosphorylated pK15.The pK15 CT fused to GST, its Y481F mutant, or GST alone were bound to glutathione beads, phosphorylated in vitro (+) or left unphosphorylated (-), and a GST pulldown was performed 32h after transfection into HEK-293T cells of expression vectors for S-tagged full-length PLCγ1 (FL) (A) or PLCγ1 SA (B) containing the indicated mutations. (C) pK15 CT WT, its Y481F mutant or GST were bound to glutathione beads, phosphorylated in vitro (+) or left unphosphorylated (-), and used for a GST pulldown assay 32h after transfecting HEK 293T cells with expression vectors for S-tagged individual PLCγ1 nSH2 or cSH2 domains.

Fig 5: The pK15 CT domain enhances Src-mediated phosphorylation of the PLCγ1 tSH2 domain.(A) The purified PLCγ1 tSH2 domain (0.6 μM) was incubated with limiting amounts (0.085 μM) of recombinant purified Src kinase expressed in insect cells in the presence of increasing concentrations (1.25–5 μM) of phosphorylated peptides representing the last 12 (pK15 12mer) or 35 (pK15 35mer) amino acids of the pK15 cytoplasmic domain for 1 hour at 37°C. The reaction was stopped using SDS sample buffer and analysed by SDS-PAGE and Western blot using an antibody to the PLCγ1 pY783 residue (top panel) or to the His tag on the recombinant tSH2 domain (bottom panel). (B) Same as above, except that a fixed concentration (5.0 μM) of the 12mer peptide, the 35mer peptide or the pK15 truncation construct GST-pK15 CTN2 [51] were used. The increased phosphorylation of the tSH2 domain in the presence of the 35mer peptide and pK15 CTN2 can be seen. (C) Increasing concentrations of the phosphorylated pK15 12mer peptide were used to inhibit the increased src-mediated phosphorylation of tSH2 induced by 2.5 μM of the pK15 35mer peptide. Experimental conditions were as in panel A.