Conceived and designed the experiments: C-HT T-HL. Performed the experiments: C-HT.
Analyzed the data: C-HT. Contributed reagents/materials/analysis tools: C-HT T-HL.
Organized the whole project and manuscript: T-HL 418
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Figure 1. Survival rates (%) of fresh water (FW)-acclimated tilapia transferred to different hypertonic conditions. Open and closed circles indicated that FW-acclimated tilapia were directly transferred to hypertonic conditions of 20‰ and 30‰ seawater (SW),
respectively. n = 30 for each experiment.
Figure 2. Confocal laser scanning micrographs of whole-mount preparations of gill filaments in tilapia at 0 h (A-C, J-L), 12 h (D-F, M-O) and 24 h (G-I, P-R) after transfer from fresh water (FW) to 20‰ seawater. Gill filaments were double-stained with anti-Na+/K+-ATPase (NKA) (red) and anti-cystic fibrosis transmembrane conductance
regulator (green; A-I) or anti-NCC/NKCC (green; J-R).
Figure 3. Dynamic changes of protein expression of heat shock protein 70 (HSP70) (A and B) and HSP90 (C and D) in gills of tilapia directly transferred from fresh water to 20‰ seawater. (A and C) Representative Western blots of HSP70 and HSP90 showed a single immunoreactive band at approximately 70 and 90 kDa,
respectively. The levels of HSP70 and HSP90 protein significantly elevated during the first 3 h post-transfer and sustained at 24 h after transfer (B and D). β-actin was used as the loading control. The asterisks indicated significant differences (P<0.05) compared with the 0 h time-point using Dunnett’s test following a one-way
ANOVA. Values are mean ± S.E.M (n = 5). M, marker.
Figure 4. Dot-blot analysis of the levels of ubiquitin-conjugated proteins in gills of tilapia 584
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directly transferred from fresh water (FW) to 20‰ seawater (SW). (A) Ubiquitin-conjugated protein levels were shown as relative values based on dot intensities.
(B) Ponceau S total protein stain of blots was used as loading control. The levels of ubiquitin-conjugated proteins increased significantly at 24 h post-transfer from FW to 20‰ SW (C). The asterisks indicated significant differences (P<0.05) compared with the 0 h time-point using Dunnett’s test following a one-way ANOVA. Values
are mean ± S.E.M (n = 5).
Figure 5. Dynamic changes of protein expression of heat shock protein 70 (HSP70) (A and B) and HSP90 (C and D) in gills of tilapia directly transferred from fresh water to 30‰ seawater (SW). (A and C) Representative Western blots of HSP70 and HSP90 showed a single immunoreactive band at approximately 70 and 90 kDa,
respectively. No significant elevation of branchial HSP70 or HSP90 was found in tilapia within 4 h post-transfer from FW to 30‰ (SW) (B and D). β-actin was used as the loading control. The asterisks indicated significant differences (P<0.05) compared with the 0 h time-point using Dunnett’s test following a one-way
ANOVA. Values are mean ± S.E.M (n = 5). M, marker.
Figure 6. Dot-blot analysis of the levels of ubiquitin-conjugated protein in gills of tilapia directly transferred from fresh water (FW) to 30‰ seawater (SW). (A) Ubiquitin-conjugated protein levels were shown as relative values based on dot intensities.
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(B) Ponceau S total protein stain of blots was used as loading control. No
significant difference was found in the levels of ubiquitin-conjugated proteins when
tilapia were transferred from FW to 30‰ SW. Values are mean ± S.E.M (n = 5).
Figure 7. The levels of aggregated proteins in gills of fresh water (FW)-acclimated tilapia acutely exposed to 20‰ (A) or 30‰ seawater (SW) (B). There was no significant difference in the levels of aggregated proteins when tilapia were transferred from FW to 20‰ SW (A). The aggregated protein levels were evidently elevated at 3 and 4 h post-transfer from FW to 30‰ SW (B). The asterisks indicated significant differences (P<0.05) compared with the 0 h time-point using Dunnett’s test
following a one-way ANOVA. Values are mean ± S.E.M (n = 5).
Figure 8. The protein abundance of branchial Na+/K+-ATPase (NKA) in aggregated protein fractions of fresh water (FW)-acclimated tilapia acutely exposed to 20‰ seawater (SW). (A) Representative Western blots of NKA revealed a single immunoreactive band at approximately 105 kDa. (B) Ponceau S total protein stain of blot. (C) There was no significant difference in the abundance of NKA in aggregated protein fractions of tilapia transferred from FW to 20‰ (SW). Values are mean ± S.E.M (n
= 5).
Figure 9. The protein abundance of branchial Na+/K+-ATPase (NKA) in aggregated protein fractions of fresh water (FW)-acclimated tilapia acutely exposed to 30‰ seawater 622
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(SW). (A) Representative Western blots of NKA revealed a single immunoreactive band at approximately 105 kDa. (B) Ponceau S total protein stain of blot. (C) The levels of NKA in aggregated fractions were significantly higher at 4 h post-transfer.
The asterisks indicated significant differences (P<0.05) compared with the 0 h time-point using Dunnett’s test following a one-way ANOVA. Values are mean ± S.E.M (n = 5).
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