Supplementary Materials

Supplementary Material for:

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief

Dane D. Jensen, TinaMarie Lieu, Michelle L. Halls, Nicholas A. Veldhuis, Wendy L. Imlach, Quynh N. Mai, Daniel P. Poole, Tim Quach, Luigi Aurelio, Joshua Conner, Carmen Klein Herenbrink, Nicholas Barlow, Jamie S. Simpson, Martin J. Scanlon, Bimbil Graham, Adam McCluskey, Phillip J. Robinson, Virginie Escriou, Romina Nassini, Serena Materazzi, Pierangelo Geppetti, Gareth A. Hicks, Macdonald J. Christie, Christopher J. H. Porter,* Meritxell Canals,* Nigel W. Bunnett*

*Corresponding author. Email: nb2733{at}cumc.columbia.edu (N.W.B.); chris.porter{at}monash.edu (C.J.H.P.); meri.canals{at}monash.edu (M.C.)

Published 31 May 2017, Sci. Transl. Med. 9, eaal3447 (2017)
DOI: 10.1126/scitranslmed.aal3447

This PDF file includes:

  • Materials and Methods
  • Fig. S1. Clathrin- and dynamin-dependent NK1R endocytosis.
  • Fig. S2. NK1R compartmentalized signaling.
  • Fig. S3. G protein–dependent NK1R signaling in endosomes.
  • Fig. S4. Nociception and inflammation in vivo.
  • Fig. S5. NK1R endocytosis in spinal neurons in vivo.
  • Fig. S6. NK1Rδ311 expression and trafficking.
  • Fig. S7. Uptake of tripartite probes.
  • Fig. S8. Effects of NK1R tripartite antagonists on ERK signaling.
  • Fig. S9. Synthesis and analysis of Span-Chol and Span–ethyl ester.
  • Fig. S10. Synthesis and analysis of L-733,060-Chol.
  • Table S1. Statistical analyses and cell replicates.
  • Legends for movies S1 to S14
  • References (3754)

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Other Supplementary Material for this manuscript includes the following:

  • Movie S1 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord slices incubated with Dy4 inact and vehicle.
  • Movie S2 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord slices incubated with Dy4 inact and SP.
  • Movie S3 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord slices incubated with Dy4 and vehicle.
  • Movie S4 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord slices incubated with Dy4 and SP.
  • Movie S5 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord 10 min after intraplantar injection of vehicle.
  • Movie S6 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord 10 min after intraplantar injection of capsaicin.
  • Movie S7 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord 10 min after intraplantar injection of capsaicin, with Dy4 injected before capsaicin.
  • Movie S8 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord 10 min after intraplantar injection of capsaicin, with Dy4 inact injected before capsaicin.
  • Movie S9 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord 10 min after intraplantar injection of capsaicin, with PS2 injected before capsaicin.
  • Movie S10 (.mp4 format). Three-dimensional projections of NK1R-IR in neurons in spinal cord 10 min after intraplantar injection of capsaicin, with PS2 inact injected before capsaicin.
  • Movie S11 (.mp4 format). Plasma membrane incorporation and endocytosis of Cy5-cholestanol by HEK293 cells.
  • Movie S12 (.mp4 format). Lack of uptake of Cy5–ethyl ester by HEK293 cells.
  • Movie S13 (.mp4 format). Time lapse images showing FRET between SNAPS-549 -NK1R Cy5-Chol.
  • Movie S14 (.mp4 format). Animation showing SP-induced assembly of endosomal signaling platform for pain transmission.