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. 2020 Mar 23;21(6):2213. doi: 10.3390/ijms21062213

Figure 3.

Figure 3

CaN inhibition permanently suppresses protein synthesis in cultured astrocytes. (A) Ctr and CaN-KO primary astrocytes were pulsed with puromycin for 3 h. Ctr were also treated with FK506 for 1 h to inhibit CaN activity. Where indicated, cycloheximide (CHX, 10 μM) was added ten minutes before adding puromycin. Anti-puromycin antibody was used to detect neo-synthesized peptides. Ponceau staining was used for band normalization. Histogram shows quantification of anti-puromycin detected bands. Data are expressed as mean  ±  SD, 5, 4, and 11 independent cultures were used for Ctr, FK506 treated and CaN-KO samples, respectively. ** p < 0.01; *** p < 0.001, one way ANOVA with Tukey post hoc test. (B) Immunofluorescence images of Ctr, Ctr treated with FK506 (200 nM) for 1, 2 and 7 days and CaN-KO, stained with anti-puromycin antibody (green). Nuclei are stained with DAPI (blue). Bar, 25 μm. Data are expressed as mean  ±  SD; * p < 0.05; ** p < 0.01, one way ANOVA with Tukey post hoc test.