Figure 4: R59 and PEST domain contribute independently to Gcm protein stability.
(a) Western blot analysis of Gcm, GcmR59L, GcmΔP, and GcmRΔP protein levels. Quantifications for relative band intensity of Flag to α-Tubulin were shown at the bottom. Western blot gels have been run under the same experimental conditions. (b) Luciferase reporter assays were done for cells transfected with p3xFlag-gcm, p3xFlag-gcmR59L, p3xFlag-gcmΔP, or p3xFlag-gcmRΔP. Note a significant difference was detected between GcmΔP and GcmRΔP. (c) ChIP analysis demonstrated that GcmΔP and GcmRΔP bind DNA. ChIP assays were performed using S2 cell lystates tranfected with the above plasmids. Each ChIP experiment was repeated three times (n = 3). (d–h”) Confocal projections of Stage 15 embryos labeled with HRP (magenta) and Repo (green) for the following genotypes: Sca-Gal4 > w1118 (d–d”, control), Sca-Gal4 > UAS-gcm (e–e”), Sca-Gal4 > UAS-gcmR59L-2 (f–f”), and Sca-Gal4 > UAS-gcmΔP (g–g”), and Sca-Gal4 > UAS-gcmRΔP (h–h”). Scale bar: 100 μm. (i) Statistics for the glial cell number per hemisegment were shown for embryos carrying the above genotypes. n = 80 (w1118 control), n = 80 (UAS-gcm), n = 35 (UAS-gcmR59L-2), n = 52 (UAS-gcmΔP), and n = 36 (UAS-gcmRΔP). Note a significant difference between embryos expressing gcm or gcmR59L with or without the PEST domain. Averages are mean ± SEM, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001 by Student’s t test between two groups and one-way ANOVA test among multiple groups. ns means no significance.
