Journal: Cell reports

Article Title: Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes.

doi: 10.1016/j.celrep.2023.113603

Figure Lengend Snippet: Figure 2. TM domain dimerization motifs do not influence EGFR diffusion (A) Mutations introduced to disrupt Sm-x-x-x-Sm dimerization motifs40 in the EGFR TM domain. The three Sm-x-x-x-Sm motifs are marked, where Sm is any small amino acid (red): T-g-m-v-G; G-m-v-g-A; and A-l-g-i-G. All Sm residues were replaced with valine in TM3X. (B) SPT data for wild-type EGFR (gray) and a variant with the TM3X TM domain shown in (A) (blue). Unliganded receptors were tracked using QD-HA (open diamonds), and ligand-bound receptors were tracked using 200 pM QD-EGF (filled diamonds). EGF induces a similar slowdown for wild-type EGFR (n = 40 cells without ligand, 42 with; p = 6.7 3 106) and the TM3X variant (n = 45 without ligand, 46 with; p = 1.9 3 107). Plots show variability and mean (±SD) across n cells. Unpaired two-sided Welch’s t tests were used to calculate p values (**p < 1 3 103; ***p < 1 3 106). See also Table S2. (C) pEGFR immunoblots of wild-type and TM3X EGFR activated with 16 nM EGF for 5 min and probed with anti-pY1173 (upper), anti-EGFR (middle), and anti-GRB2 (lower) as loading control. Representative of three biological re- peats.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER N-terminally HaloTagged EGFR(DC-Tail) in pcDNA3.1 This study N/A pHTC HaloTag CMV-neo Vector Promega Cat#: G7711 pFastbac Vector Kit ThermoFisher Scientific Cat#: 10360014 pFastbac sEGFR Ferguson et al.3 N/A pFastbac EGFR TKD [672–998] Park et al.59 N/A pFastbac EGFR JM-TKD [645–998] Red Brewer et al.9 N/A pFastbac EGFR JM-TKD [645–998], V924R This study N/A pFastbac EGFR TKD [672–998], I682Q This study N/A pSpCas9(BB)-2A-GFP (PX458) Ran et al.74 Addgene Cat#: 48138 Software and algorithms DBSCAN Ester et al.75 N/A SMITE (Single Molecule Imaging Toolbox Extraordinaire) K. Lidke lab76 https://github.com/LidkeLab/smite MATLAB version: 9.13.0 (R2022b) The MathWorks Inc., Natick, MA, USA https://www.mathworks.com Origin 2021b (9.8.5.201) OriginLab Corporation, Northampton, MA, USA https://www.originlab.com/ PyMol Molecular Graphics System (Version 2.5.0) Schrödinger, LLC http://www.pymol.org GraphPad Prism 9.5.1 GraphPad Software, La Jolla California USA https://www.graphpad.com/ ExPASy ProtParam Tool Gasteiger et al.77 https://web.expasy.org/protparam/ Broad Institute gRNA designer Broad Institute https://portals.broadinstitute.org/gppx/ crispick/public Adobe Photoshop 24.2.0 Adobe Inc. https://www.adobe.com/products/ photoshop.html

Techniques: Diffusion-based Assay, Variant Assay, Western Blot, Control