FIGURE 4:

Depletion of Fign increases tyr-MTs in the cortical region of cultured astrocytes. (A) Left, representative images of α-tubulin (green)– and tyr-tubulin (red)–expressing astrocytes treated with Ctrl siRNA and Fign siRNA. Compared to Ctrl group, depletion of Fign resulted in MTs curved in cell cortex regions, and these MTs are mainly composed of tyr-tubulin. Bar, 20 μm. Right, top, quantification of the relative fluorescence intensity of tyr-tubulin protein. Compared to the Ctrl siRNA–treated cells, Fign siRNA increased tyr-tubulin mass. The data are shown as mean ± SE. The relative fluorescence intensity of Ctrl siRNA–treated astrocytes is normalized as 1. Fign siRNA = 1.42 ± 0.14, n = 18, *p < 0.05. (B) Representative Western blotting result. Top, Fign siRNA treatment increased the ratio of tyr-tubulin compared with Ctrl siRNA treatment for 3 d. Bottom, tyr-tubulin protein increased significantly after Fign knockdown. The data are presented as mean ± SE. The relative tyr-tubulin protein level (α-tubulin as the control) of Ctrl siRNA–treated astrocytes was normalized as 1. Fign siRNA = 1.46 ± 0.22, n = 4, *p < 0.05. (C) Top, astrocytes treated with Ctrl or Fign siRNA for 3 d, fixed, and immunostained for detyrosinated or acetylated tubulin. To further illustrate that Fign depletion reduced tyr-tubulin mass, detyrosinated and acetylated tubulins were chosen as the controls. Bar, 20 μm. Bottom, quantification, with relative fluorescence intensity of Ctrl siRNA–treated astrocytes normalized as 1. There were no significantly different alterations for detyrosinated tubulin (bottom, left; Fign siRNA = 1.09 ± 0.07) or acetylated tubulin (bottom, right; Fign siRNA = 1.14 ± 0.06). n = 30; ns, not significant; p > 0.05.