Supplementary Materials

Supplementary Materials for:

De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling

Lars Philipsen, Amarendra V. Reddycherla, Roland Hartig, Janine Gumz, Matthias Kästle, Andreas Kritikos, Mateusz P. Poltorak, Yury Prokazov, Evgeny Turbin, André Weber, Werner Zuschratter, Burkhart Schraven,* Luca Simeoni,* Andreas J. Müller*

*Corresponding author. Email: andreas.mueller{at}med.ovgu.de (A.J.M.); luca.simeoni{at}med.ovgu.de (L.S.); burkhart.schraven{at}med.ovgu.de (B.S.)

This PDF file includes:

  • Methods
  • Fig. S1. Efficiency of the reconstitution of TCR-proximal signaling in T cells by transient transfection with plasmids encoding fluorescently tagged Lck variants.
  • Fig. S2. Comparison of the conformational dynamics of the Y394A and Y394F single mutant and the Y394A/Y505F and Y394F/Y505F double mutant Lck biosensors.
  • Fig. S3. Influence of mutating Tyr394 or Tyr505 on the abundance of TqLck-V2 in transiently transfected JCaM 1.6 cells.
  • Fig. S4. Capability of the wild-type and tyrosine-mutated TqLck-V2 biosensors to reconstitute TCR-proximal signaling.
  • Fig. S5. An optimized staining protocol for investigating Lck at the single-cell level.
  • Fig. S6. Influence of T cell activation by mouse anti-CD3 IgG1 on the detection of Lck with a primary mouse IgG2b antibody against total Lck.
  • Fig. S7. Analysis of Tyr505 phosphorylation upon TCR stimulation of JCaM 1.6 cells expressing constitutively closed TqLck-V2 biosensors.
  • Table S1. Comparison of the ECFP and Turquoise2 FRET donors and of the ECFP-EYFP and Tq-V2 FRET pairs.

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Citation: L. Philipsen, A. V. Reddycherla, R. Hartig, J. Gumz, M. Kästle, A. Kritikos, M. P. Poltorak, Y. Prokazov, E. Turbin, A. Weber, W. Zuschratter, B. Schraven, L. Simeoni, A. J. Müller, De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling. Sci. Signal. 10, eaaf4736 (2017).

© 2017 American Association for the Advancement of Science