Extended Data Figure 8. Effect of expression of phosphomimetic alpha-actinin 1^Y246Eon F-actin organization and TRIM21 sequestration.

a, TIRF microscopy of TRIM21-GFP and F-actin in NSCLCs. Scale bar, 10 μm. Representative images from a single imaging experiment. b, Fluorescence microscopy of TRIM21-GFP and F-actin in HBEC76 treated with lysophosphatidic acid (LPA; 20 μM) for 30 min. Scale bar, 10 μm. Representative images from a single imaging experiment. c, Epi-fluorescence microscopy of HBEC76 expressing GFP alone, wildtype alpha-actinin 1 (ACTN1)-GFP or mutant alpha-actinin 1^Y246E (ACTN1^Y246E)-GFP. Scale bar, 10 μm. Representative images from a single imaging experiment. d, Epi-fluorescence microscopy of HEK cells expressing GFP alone, ACTN1-GFP or mutant ACTN1^Y246E-GFP. Scale bar, 10 μm. Representative images from a single imaging experiment. e, Epi-fluorescence microscopy of TRIM21-GFP and F-actin following immunofluorescence labeling of Flag-tagged wildtype α-actinin 1 (WT ACTN1) or α-actinin 1 harboring the Y246E mutation (ACTN1^Y246E; the cell is outlined with a dotted yellow line) in HBEC76. Scale bar, 10 μm. Representative images from three independent experiments. f, Epi-fluorescence microscopy of TRIM21-GFP and F-actin upon over-expression of wildtype α-actinin 1 (WT ACTN1) or mutant α-actinin 1 (ACTN1^Y246E) in HEK cells. Scale bar, 10 μm. Representative images from a single imaging experiment. g, Abundance of PFKP on stiff and soft substrates upon over-expression of GFP alone, ACTN1-GFP or mutant ACTN1^Y246E-GFP in HEK cells. Representative data from two independent experiments. Areas of zoom-in are indicated by red boxes (a, b). In all images (a-f), F-actin was stained with fluorescently conjugated phalloidin. Protein abundance was normalized by GAPDH (g).