*4.9. Two-Dimensional Electrophoresis*

Two-dimensional electrophoresis was performed as previously described [9,10]. Untreated *S. costatus* cells and exposed to 0.4 M NaCl stress under high light conditions were harvested and promptly frozen in liquid nitrogen. Proteins of several samples were sedimented by acetone, and the protein pellet was dried in vacuo. Dried pellet was dissolved in a two-dimensional (2D) electrophoresis sample buffer containing 8 M urea, 30 mM DTT, 2% (*v*/*v*) Pharmalyte 3–10 (Pharmacia), and 0.5% Triton X-100. Both isoelectric focusing (IEF) and SDS-PAGE were performed horizontally with Maltiphor II (Pharmacia, Peapark, NJ, USA). For electrophoresis, 40 μg of total protein was loaded onto gels. For detection, the gels were silver-stained with a Silver Staining Kit (Invitrogen).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/md19060349/s1, Figure S1: The deduced amino acid sequences of the AstaP orthologs. Figure S2: Comparison of amino acid sequences among AstaP-proteins in *C. vacuolata.*

**Author Contributions:** Conceptualization, H.T., A.M., and S.K.; methodology, H.T., A.M., R.Y., S.T., and S.K.; investigation, H.T., A.M., R.Y., T.I., and S.K.; data curation, H.T., and S.K.; writing—original draft preparation, H.T., and S.K.; writing—review and editing, H.T., S.T., and S.K.; visualization, H.T. and S.K.; funding acquisition, S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported in part by a grant-in-aid from the Japan Society for the Promotion of Science (No. 26440155), the Institute for Fermentation, Osaka (IFO, G-2014-2-072), and the Cosmetology Research Foundation (J-15-2) (to SK), and MEXT-Supported Program for the Strategic Research Foundation at Private Universities (S1311017).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The cDNA sequence data of CvacAstaP1, CvacAstaP2, and ScosAstaP have been deposited in the NCBI database under the accession numbers MZ063685–MZ063687, respectively.

**Acknowledgments:** The authors thank many colleagues for advice, discussion, and technical assistance, especially Keita Yamazaki, Natsumi Abe, Ayae Furukawa, Takumi Satoh, and Youichi Niimura. The authors thank Eri Kubota for performing RNA sequencing.

**Conflicts of Interest:** The authors declare no conflict of interests.
