Figure EV5. Cortical actin tension modulates plasma membrane protein composition.

• A
  Excess chemical potential required for the recruitment of positive curvature sensing domains in basal, annulus, and protrusion regions of the plasma membrane plotted as a function of A/Ap.
• B
  Immunoblots of Exo70‐GFP and alpha‐tubulin (control) in MCF10A MECs ligated with rBM in 2D or 3D. Quantification of the levels of plasma membrane‐proximal Exo70 at different focal planes (apical, middle, and basal) in MCF10A MECs ligated with rBM in 2D or 3D. The levels of plasma membrane‐proximal Exo70 in MECs were quantified as plasma membrane fluorescence (GFP colocalization with farnesylated mCherry) relative to total cellular Exo70‐GFP fluorescence (mean ± SEM; 2D, n = 22; 3D, n = 23 cells from two independent experiments). Statistical analysis by one‐way ANOVA followed by Uncorrected Fisher's LSD. Apical and middle 2D versus 3D, ****P < 0.0001; basal 2D versus 3D, ***P = 0.0007.
• C
  Representative fluorescence microscopy images of MCF10A MECs stably expressing recombinant V5‐APEX2‐CAAX and ligated to rBM in 2D in the absence and presence of blebbistatin. MECs were immunostained with antibodies targeting V5 (green) and counterstained with phalloidin (red). Scale bar, 10 μm.
• D
  MECs ligated to rBM in 2D with and without 2 h of blebbistatin treatment were harvested for immunoblotting. The activity of APEX2‐CAAX (V5) was examined via immunoblot for biotinylated proteins with streptavidin‐HRP.
• E
  Schematic of the strategy used for the two‐state SILAC experiment. MECs expressing APEX‐CAAX were treated with biotin‐phenol overnight followed by 1 min of H₂O₂ exposure. MECs labeled with heavy isotope amino acids were treated with blebbistatin to reduce myosin II activity, whereas those labeled with light amino acids were treated with DMSO (vehicle). Cells were lysed and excess biotin phenol trapped in the polyacrylamide gels was removed using acetone precipitation. The resuspended protein was purified using streptavidin beads and identified by mass spectrometry. For each protein, the H/L SILAC ratio reflects the extent of its biotinylation by APEX2‐CAAX in the presence/absence of blebbistatin.
• F
  GO Cellular Component analysis of proteins enriched at the plasma membrane of cells with low cortical tension (Fig 7I) using PANTHER online database.