Fig 2.

 Effect of ectoine treatment on heat shock protein (Hsp)70B′ gene expression in keratinocyte cells. (A) Reverse transcriptase– polymerase chain reaction analysis using specific primers for hsp70B′ messenger RNA (mRNA) expression. Lane 1: untreated-cell mRNA; lanes 2–5: mRNA from keratinocytes pretreated for 24 hours with 50, 100, 250, 500 μg/mL ectoine, respectively, not heat stressed; lane 6: mRNA from heat-stressed keratinocytes; lanes 7–10: mRNA from keratinocytes pretreated for 24 hours with 50, 100, 250, 500 μg/mL ectoine, respectively, and, successively, heat stressed. The hsp70B′:β-actin fluorescence intensity ratios were as follows: lanes 6, 7, 8, 9, and 10, 0.35 ± 0.12, 0.45 ± 0.18, 0.87 ± 0.11, 0.43 ± 0.10, and 0.39 ± 0.21, respectively. The values are the mean of at least 5 independent experiments ± SD (P < 0.05). (B) Western blot analysis for the hsp70B′ content. Fifty micrograms of cell lysates from each sample was separated by a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and subjected to Western blot analysis using 1 μg/mL anti-hsp70B′ polyclonal antibody as a probe