Fig 2: Integrin adhesion complexes are sensitive to genetic perturbations that alter Cas-dependent phase separation.(A) In vitro solution turbidity measurements. 500 nM Nck and 1 µM N-WASP were combined with increasing concentrations of phosphorylated Cas (pCas, magenta) or Y15F Cas (black). Each point represents the mean ± SEM of three independent measurements. (B) Representation of in vitro experiments in C-D. (C) Spinning disk confocal fluorescence microscopy images of droplets formed with the Cas + FAK mix. TOP: 1 µM each of pCas, Nck, N-WASP, Kin, FAK, and paxillin (15% Alexa546); BOTTOM: 1 µM each of Y15F Cas, Nck, N-WASP, Kin, FAK, and paxillin (15% Alexa546). (D) Quantification of paxillin partitioning into droplets. Each condition contains at least 40 measurements from two independent experiments. (E) Spinning disk fluorescence microscopy images of MEFs with immunostaining for endogenous paxillin. (F) Quantification of number of adhesions. (G) Quantification of paxillin partitioning into adhesions (partition coefficient). Significance tested by one-way ANOVA followed by a Tukey multiple comparison test. In (C) scalebar = 5 µm. In (E) scalebar = 10 µm. Note that the pCas protein used in Figure 6 is a different batch than protein used in Figures 1—5 (See Figure 1—figure supplement 3).

Fig 3: Cas and FAK synergistically promote nascent adhesion assembly during cell spreading.(A) Western blot analysis of FAK knockdown. WT (+) or Cas knockout (-) MEFs were treated with nontargeting (+) or FAK (si) siRNA for 48 hr and lysates were blotted with Cas, FAK or actin antibodies. (B) Total Internal Reflection Fluorescence (TIRF) microscopy images of MEFs fixed after 5 min of spreading with immunostaining for endogenous paxillin. Scalebar = 5 µm. (C) Quantification of number of adhesions. Each grey point represents a measurement from one cell, and the mean ± SEM mean is indicated by black lines. Data from at least 60 cells from two or more independent experiments. Significance tested by one-way ANOVA followed by a Tukey multiple comparison test. Figure 8—source data 1.Uncropped gel images and unprocessed gel.tif files from Figure 8a.

Fig 4: Downstream targets of Src in mouse and cellular models of SBMA. a–c Immunoblots showing the levels of phosphorylated and total p130Cas, Stat3, p38MAPK, and Akt proteins in the spinal cord and skeletal muscle of 9-week-old AR-97Q mice treated with or without A419259 (a). Quantitative analyses were performed using densitometry (b, c). d–f Immunoblots showing the levels of the phosphorylated and total Src, p130Cas, Stat3, p38MAPK, and Akt proteins in DHT-treated NSC34 and C2C12 cells stably expressing AR-97Q with or without 0.2 µM of A419259 administration (d). Quantitative analyses were performed using densitometry (e, f). g–i Immunoblots showing the levels of the phosphorylated and total Src, p130Cas, Stat3, and p38MAPK proteins in the NSC34 and C2C12 cells transfected with mock or Src plasmids (g). Quantitative analyses were performed using densitometry (h, i). Quantitative analyses were performed on samples from n = 3 animals per group. Error bars indicate the s.e.m. Statistical analyses were performed using the unpaired two-sided t-test (b, c, e, f, h, i). *p < 0.05 and **p < 0.01. N.S., not significant. Source data are provided as a Source Data file (a–i)

Fig 5: p130Cas is activated in cellular and mouse models of SBMA. a, b Immunoblots showing the levels of the phosphorylated and total p130Cas proteins in NSC34 cells and C2C12 cells stably expressing AR-24Q or AR-97Q that were treated with or without DHT (a). Quantitative analyses were performed using densitometry (n = 3) (b). c–e Immunoblots showing levels of the p-p130Cas and p130Cas proteins in the spinal cord and skeletal muscle of wild-type and AR-97Q mice at 6, 9 and 13 weeks of age (c). Quantitative analyses were performed using densitometry (n = 3) (d, e). f, g Immunohistochemistry for p-p130Cas in spinal cord (f) and skeletal muscle (g) samples from 6-week-old mice. Error bars indicate the s.e.m. Statistical analyses were performed using the unpaired two-sided t-test (b, d, e). *p < 0.05 and **p < 0.01. Wt, wild-type; AR, AR-97Q; N.S., not significant. Scale bars: 25 µm (f, g). Source data are provided as a Source Data file (a–e)