Journal: Nature Communications

Article Title: Covalent modification of a glutamic acid inspired by HaloTag technology

doi: 10.1038/s41467-026-68999-9

Figure Lengend Snippet: a mCitrine lifetime (τ) measurement in HEK293T cells with mCitrine-Rheb only, or donor-acceptor system with mCitrine-Rheb bound to mCherry-PDEδ, or donor-acceptor system with DeltaTag treatment for 10 min. First column: fluorescence intensity distribution mCitrine-Rheb; second column: zoomed-in views for cellular mCitrine-Rheb distribution; third column: mCitrine-PDEδ; fourth column: global lifetime distribution; fifth column: lifetime decay of mCitrine and fitting with mono-exponential decay. Scale bar, 25 µm. Images represent biological replicates n = 3 (each with technical replicates N = 6). b Average lifetime of mCitrine. Data are presented as mean ± s.d., representative of biological replicates n = 3. Unpaired t -test, two-tailed p -value = 0.0036 (**). c Immunofluorescence staining of PA-TU-8902 cells with anti-Rheb antibody, overnight after treatment, representative of biological replicates n = 3. Intensity profiles (arbitrary unit, A.U.) along the lines are plotted against distance (µm). Scale bars, 20 µm. d Plot of kinase z -scores (two-tailed p -value ≤ 0.05) after kinase-substrate enrichment analysis (KSEA App with NetworKIN, substrate count cutoff = 2, NetworKIN score cutoff = 1) – of significant hits from phosphoproteome profiling upon treatment of 6a (see Supplementary Fig. for details). e Reactome pathway overrepresentation of downregulated kinases, with Voronoi visualisation zooming into mTOR signalling. The scale of colour intensity is an indication of the p -value of pathway overrepresentation. f Western blot analysis of S6P phosphorylation on Ser235 and Ser236 (S235/S236) and total S6P (tS6P) in PA-TU-8902 cells. (−/−) represents unstimulated DMSO-treated cells. Quantification of per cent pS6P/tS6P ± s.d. was normalised to EGF-stimulated DMSO control at each time-point, representative of biological replicates n = 3. Unpaired t -test, two-tailed p -values comparing each condition to EGF-stimulated DMSO control (−/+): 3 h: vs 1 , p = 0.78; vs 6a , p = 0.035 (*); 5 h: vs 1 , p = 0.138; vs 6a , p = 0.0009 (**); 8 h: vs 1 , p = 0.102; vs 6a , p < 0.0001 (***); p -value > 0.05 are labelled as non-significant (ns). j Schematic representation of inhibition along the PDEδ-Rheb-mTORC1 axis, created in BioRender. Zhang, R. (2026) https://BioRender.com/dxvyoxr . Source data are provided as a Source Data file.

Article Snippet: Cells were subsequently blocked by 2% BSA in PBS-T (0.1% Tween 20 in PBS) for 1 h at room temperature, incubated with the primary antibody Anti-Rheb (Santa Cruz Biotechnology Cat# sc-271509, RRID: AB_10659102, 1:500) overnight at 4 °C in blocking buffer, then washed with PBS-T three times and incubated with Alexa-555-conjugated secondary antibody (Invitrogen # A-31570, RRID:AB_2536180, 1: 1000) and DAPI (1 μg/ml) in blocking buffer for 1 h at room temperature with protection from light.

Techniques: Fluorescence, Two Tailed Test, Immunofluorescence, Staining, Western Blot, Phospho-proteomics, Control, Inhibition