Figure 6. α -Actin overexpression... | Rockefeller University Press

Figure 6.

$Figure 6. α-Actin overexpression restores the impaired F-actin content of Srf-deleted MBs. (A) Staining for F-actin (phalloidin) and nuclei (DAPI) on control, Srf-deleted (Mut), and Srf-deleted MBs overexpressing α-actin (Mut/Act+). (B) Quantification of F-actin by measuring the total phalloidin fluorescence intensity per cell (ImageJ) in control, mutant, and Mut/Act+ MBs (one representative experiment). (C) Representative immunoblot showing total actin (pan-Actin) in control, mutant, and Mut/Act+ MBs. Tubulin was used as a loading control. (D) Quantification of the pan-actin/tubulin ratio from immunoblots (n = 5–9). (E) Representative immunoblot showing actin in the insoluble (F) versus soluble (G) fractions in control, mutant, and Mut/Act+ MBs. (F) Quantification of the F-/G-actin ratio from immunoblots (n = 6–8). (G) Representative confocal projections of z-sections of F-actin staining (phalloidin) taken from the adherent (ventral) cell bottom and middle and proceeding up to the media-facing top of control, mutant, and Mut/Act+ MBs. (H) Survey view of the cytoplasmic surface of the plasma membrane from unroofed control MBs. (H′) Higher-magnification view from H. (H″) Higher-magnification view corresponding to the boxed regions in H′. (I) Survey view of the cytoplasmic surface of the plasma membrane from unroofed mutant MBs. (I′) Higher-magnification view from I. (I″) Higher-magnification view corresponding to the boxed regions in I′. (J) Survey view of the cytoplasmic surface of the plasma membrane from unroofed Mut/Act+ MBs. (J′) Higher-magnification view from J. (J″) Higher-magnification view corresponding to the boxed region in J′. Data are mean ± SEM. *, P < 0.05; **, P < 0.01.$

α-Actin overexpression restores the impaired F-actin content of Srf-deleted MBs. (A) Staining for F-actin (phalloidin) and nuclei (DAPI) on control, Srf-deleted (Mut), and Srf-deleted MBs overexpressing α-actin (Mut/Act⁺). (B) Quantification of F-actin by measuring the total phalloidin fluorescence intensity per cell (ImageJ) in control, mutant, and Mut/Act⁺ MBs (one representative experiment). (C) Representative immunoblot showing total actin (pan-Actin) in control, mutant, and Mut/Act⁺ MBs. Tubulin was used as a loading control. (D) Quantification of the pan-actin/tubulin ratio from immunoblots (n = 5–9). (E) Representative immunoblot showing actin in the insoluble (F) versus soluble (G) fractions in control, mutant, and Mut/Act⁺ MBs. (F) Quantification of the F-/G-actin ratio from immunoblots (n = 6–8). (G) Representative confocal projections of z-sections of F-actin staining (phalloidin) taken from the adherent (ventral) cell bottom and middle and proceeding up to the media-facing top of control, mutant, and Mut/Act⁺ MBs. (H) Survey view of the cytoplasmic surface of the plasma membrane from unroofed control MBs. (H′) Higher-magnification view from H. (H″) Higher-magnification view corresponding to the boxed regions in H′. (I) Survey view of the cytoplasmic surface of the plasma membrane from unroofed mutant MBs. (I′) Higher-magnification view from I. (I″) Higher-magnification view corresponding to the boxed regions in I′. (J) Survey view of the cytoplasmic surface of the plasma membrane from unroofed Mut/Act⁺ MBs. (J′) Higher-magnification view from J. (J″) Higher-magnification view corresponding to the boxed region in J′. Data are mean ± SEM. *, P < 0.05; **, P < 0.01.