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. Author manuscript; available in PMC: 2014 Aug 5.

Published in final edited form as: Mech Ageing Dev. 2014 May 9;139:1–10. doi: 10.1016/j.mad.2014.04.001

Fig. 3 — A. HSR in D. pulex isolate TCO. B: HSR in D. pulex isolate RW20. C. HSR in D. pulicaria isolate 3L2-1. D. HSR in D. pulicaria isolate lake XVI-clone 11. Daphnia were subjected to heat shock at 32° C for 30 minutes, allowed to recover for indicated time periods, and protein was extracted. Western Blot analysis was performed using 50 μg of total protein with anti-Hsp70 antibody. The recovery periods after heat shock are as indicated above the lanes and the age of the Daphnia are indicated in weeks below the panels. Blots were re-probed with anti-α tubulin antibody to ensure even loading. E-F. Quantification of western blot data in Figs. 3 A-D: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from several independent biological replicates (3 replicates for pulex ecotypes and 4 replicates for pulicaria ecotypes) is represented as bar graphs and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes and all Hsp70 signals were normalized to band intensities of β-actin for each lane. The black bars represent control samples, the white bars represent samples after 4 h recovery, and grey bars represent samples after 6 h recovery. As shown above bars, a, d, g, h, j and k labels indicate P values that show a significant difference (0.0010, 0.0018, 0.0014, 0.0008, 0.0021, and 0.0038 resp.) compared to controls. The P values indicated by labels b, c, e, f, i, and l exhibited no significant difference (0.034, 0.045, 0.086, 0.052, 0.11, and 0.21) compared to controls.There was no significant difference observed in signals for β-actin in various lanes with significant values being < 0.01.

Fig. 3 — A. HSR in D. pulex isolate TCO. B: HSR in D. pulex isolate RW20. C. HSR in D. pulicaria isolate 3L2-1. D. HSR in D. pulicaria isolate lake XVI-clone 11. Daphnia were subjected to heat shock at 32° C for 30 minutes, allowed to recover for indicated time periods, and protein was extracted. Western Blot analysis was performed using 50 μg of total protein with anti-Hsp70 antibody. The recovery periods after heat shock are as indicated above the lanes and the age of the Daphnia are indicated in weeks below the panels. Blots were re-probed with anti-α tubulin antibody to ensure even loading. E-F. Quantification of western blot data in Figs. 3 A-D: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from several independent biological replicates (3 replicates for pulex ecotypes and 4 replicates for pulicaria ecotypes) is represented as bar graphs and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes and all Hsp70 signals were normalized to band intensities of β-actin for each lane. The black bars represent control samples, the white bars represent samples after 4 h recovery, and grey bars represent samples after 6 h recovery. As shown above bars, a, d, g, h, j and k labels indicate P values that show a significant difference (0.0010, 0.0018, 0.0014, 0.0008, 0.0021, and 0.0038 resp.) compared to controls. The P values indicated by labels b, c, e, f, i, and l exhibited no significant difference (0.034, 0.045, 0.086, 0.052, 0.11, and 0.21) compared to controls.There was no significant difference observed in signals for β-actin in various lanes with significant values being < 0.01.