Research Paper Volume 14, Issue 7 pp 2989—3029

Figure 6. Expression of heat shock genes during aging in flies expressing NtFT4. Relative expression of two small heat shock protein genes directly associated with aging (Hsp26 and Hsp27) (A), of the two small heat shock protein genes Hsp22 and Hsp23 (B), of larger heat shock protein genes Hsp83, HspB8, Hsc70-3 and Hsc70-4 (C), and of weakly-expressed heat shock protein genes DnaJ-1, l(2)efl, Hsp68 and Hsp70Aa (D) in female da > NtFT4 flies aged 10, 20 and 50 d, compared with da-Gal4 flies by quantitative RT-PCR. Relative expression was calculated using Gapdh2 as a reference gene. Data are means ± SEM (n = 3). Significance was tested by one-way ANOVA and Tukey’s post hoc test for changes during age (10 d vs. 20 d vs. 50 d) and using a t-test for pairwise comparisons between da-Gal4 and da > NtFT4 flies (^***p < 0.01, ^**p < 0.05, ^*p < 0.1, Abbreviation: NS: not significant; a = significant between 10 d and 20 d, b = significant between 10 d and 50 d, c = significant compared between 20 d and 50 d). (E) Western blot showing the detection of HSP26 in protein extracts from female da > NtFT4 flies aged 10, 20 and 50 d, compared with da-Gal4 flies. A representative Western blot is shown for anti-HSP26 and comparable protein loading was ensured by staining with Coomassie Brilliant Blue. (F) Quantification of relative band intensities from three independent Western blot samples from 20 d (highest levels of HSP26 protein) and 50 d old flies. The relative band intensity was measured with imageJ and calculated by referring to the weakest band on each blot (50 d old da-Gal4 flies). Data are means ± SEM (n = 3), p = 0.029 (t-test), Abbreviation: NS: not significant. The p-values of all comparisons are provided in Supplementary Table 9.