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. 2021 Dec 13;118(51):e2108104118. doi: 10.1073/pnas.2108104118

Fig. 6.

Fig. 6.

Specific interaction between WA and WB chains of tiger salamander. (A–D) Production of recombinant WA and WB proteins in transfected cells. Total proteins from Chinese hamster ovary (CHO) K-1–transfected cells used for flow cytometry in E and F were investigated by Western blot analyses. Above each lane, two kinds of cotransfected DNA are indicated. (A) FLAG-tagged WA detected by anti-FLAG antibody. (B) PA-tagged WB detected by anti-PA antibody. Apparent molecular weights of protein-size markers are indicated in kilo Dalton. Abbreviations are the following: pFLAG, empty FLAG vector; pPA, empty PA vector; TS FLAG-WA, tiger salamander N-terminal FLAG-tagged WA; TS PA-WB, tiger salamander N-terminal PA-tagged WB; MT PA-WB, Mexican tetra (teleost fish) N-terminal PA-tagged WB. MT PA-WB was used instead of TS PA-WB in some cases as a recombinant WB of a distantly related species (see Fig. 1 for tiger salamander and Mexican tetra). β-actin controls using the membrane in A or B are shown in SI Appendix, Fig. S13. (C and D) Digestion of recombinant WA protein by Endoglycosidase H (Endo H), which does not cleave highly processed complex oligosaccharides, or Glycopeptidase F (GPF), which broadly cleaves oligosaccharides from N-linked glycoproteins. Enzyme-digested total proteins from transfected CHO-K1 cells were investigated by Western blot analyses. (C) FLAG-tagged WA with/without (±) Endo H digestion detected by anti-FLAG antibody. (D) FLAG-tagged WA with/without (±) GPF digestion detected by anti-FLAG antibody. The results of the PA-tagged WB are shown in SI Appendix, Fig. S14. Apparent molecular weights of protein-size markers are indicated in kilo Dalton. See SI Appendix for details. (E and F) Cell surface expression of recombinant WA and WB proteins of tiger salamander in CHO K-1–transfected cells, as determined by flow cytometry. Above each panel, two kinds of cotransfected DNA are indicated. (E) Anti-FLAG antibody binding to N-terminal FLAG-tagged WA on the cell surface. Solid lines represent the results with anti-FLAG antibody, and gray lines represent the results with isotype control antibody. (F) Anti-PA antibody binding to N-terminal PA-tagged WB on the cell surface. Solid lines represent the results with anti-PA antibody, and gray lines represent the results with isotype control antibody. Abbreviations are the same as those in A–D. See SI Appendix for details.