1. Confirmation of cytosolic localized Immunoglobulin structure in the reducing environment of the mammalian cell cytosol 2015. 05. 20 Lee yeonjin

2. GENETIC BASIS OF ANTIBODY DIVERSITY BY, REKHA.M.S Antibody secretion pathway

3. Protein folding and formation of disulfides are thus favored in this oxidizing environment. Protein disulfide isomerase (PDI) plays an important role in these activities. Protein disulfide isomerase, a catalyst in the oxidative folding, is not present in the cytosol. These compartments contain sulfhydryl oxidase enzymes that catalyze the pairing and oxidation of cysteine residues. In contrast, most proteins in a healthy cytosol are maintained in reduced form through surveillance by NADPH-dependent reductases and the lack of sulfhydryl oxidases. J Biol Chem. 2002 May 31;277(22):19339-45. Epub 2002 Mar 26. Adv Drug Deliv Rev. 2003 Feb 10;55(2):199-215. ANTIOXIDANTS & REDOX SIGNALING Volume 13, Number 8, 2010

4. Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event. The cytoplasm and the nucleus are examples of reducing environments in which most proteins are (at least partially) in a reduced form. Cytosolic and nuclear proteins generally contain few disulfide bonds. The reducing environment of the cytoplasm prevents the formation of conserved interchain bridges. J Control Release. 2014 Dec 10;195:147-54. doi: 10.1016/j.jconrel.2014.06.012. Epub 2014 Jun 18. Sci STKE. 2004 Jun 22;2004(239):pe27. FEBS Lett. 2001 Nov 23;508(3):407-12.

5. Genes encoding the heavy and light chains of a hapten-specific IgM antibody were modified by site-directed mutagenesis to destroy the hydrophobic leader sequences and allow expression in the cytoplasm of non-lymphoid cells. The in situ assembly of the mutant heavy and light chains was tested in transfected cell lines by immunofluorescence using anti-idiotypic antibodies. A positive diffuse cytoplasmic staining was observed. This demonstrated that the antibody polypeptide chains could assemble in the cell cytoplasm. We demonstrate that a scFv can form intramolecular disulfide bridges and is functionally expressed in the cytosol of stably transformed plants. In addition, the formation of intermolecular disulfide bridges through a cysteine present in the linker peptide was observed. The EMBO Journal vol.9 no. 1 pp.101 - 108, 1990 J Biol Chem. 2002 May 31;277(22):19339-45. Epub 2002 Mar 26.

6. One question is how substrates of cytosolic sulfhydryl oxidases remain oxidized in the presence of competing systems for disulfide reduction. On the first level, burial of disulfides in the cores of viral proteins would protect them from cytosolic reductants. On the second level, segregation of viral protein production and folding from bulk cytosol may help to exclude proteins that catalyze disulfide reduction. Antioxid Redox Signal. 2010 Oct;13(8):1261-71. doi: 10.1089/ars.2010.3128.

7. Construction of a plasmid to express the IgG protein in mammalian cell

8. Confirmation of antibody structure in cell cytosol using Western blot Reducing Non-Reducing IB : Human IgG- Fc Cytosolic expressed IgG 50 kDa 150 kDa or Experiment design

9. Confirmation of antibody structure in cell cytosol using Western blot kDa ΔLd ch3D8 IgG 6C407 IgG 9C353 IgG 50― 25― IB : Human IgG-Fc IB : Human IgG-kappa KV10 originonly HeLa cell REDUCING CONDITION HeLa cell

10. Confirmation of antibody form in cell using Western blot NON-REDUCING CONDITION HeLa cell ΔLd KV10 origin only HeLa cell ch3D8 IgG 6C407 IgG 9C353 IgG IB : Human IgG-Fc kDa 75― 50― 100― 140―

11. Confirmation of antibody structure in cell cytosol using O2F3 IgM ※ O2F3 IgM – Anti-3D8 idiotypic antibody IMMUNOLOGICAL INVESTIGATIONS Vol. 31, Nos. 3 & 4, pp. 205–218, 2002

12. ① ② ③ Protein A ① ② ③ ① Goat anti-human IgG (Fc specific) 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Rabbit anti-human kappa (1:2000) ④ Goat anti-rabbit IgG (Fc specific)-AP conjugated (1:5000) ① ② ③ ④ ① Protein L coating 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Goat anti-human IgG (Fc specific)-AP conjugated (1:5000) ① ② ③ Protein L ① Protein A coating 1 μg/ml ② Cellulary expressed DNA cell lysate ③ F(ab’)2-Goat anti-human kappa-AP conjugated (1:5000) ① Rabbit anti-human kappa 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Goat anti-human IgG (Fc specific)-AP conjugated (1:5000)

13. Confirmation of antibody structure in cell cytosol using ELISA ① Goat anti-human IgG (Fc specific) 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Rabbit anti-human kappa (1:2000) ④ Goat anti-rabbit IgG (Fc specific)-AP conjugated (1:5000) ① ② ③ ④ ① Protein L coating 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Goat anti-human IgG (Fc specific)-AP conjugated (1:5000) ① ② ③ Protein L Experiment design

14. Confirmation of antibody structure in cell cytosol using ELISA ΔLd kDa KV10 origin only HeLa cell ch3D8 IgG 50― 25― IB : Human IgG-Fc IB : Human IgG-kappa 10 ng 5 ng 1 ng Purrified ch3D8 IgG Goat anti-human IgG(Fc specific) #52 (1:3000) Goat anti-human Kappa chain #148 (1:3000) Rabbit anti-goat IgG(H+L)-HRP #56 (1:3000)

15. Confirmation of antibody structure in cell cytosol using ELISA ① Protein A coating 1 μg/ml ② Cellulary expressed DNA cell lysate ③ F(ab’)2-Goat anti-human kappa-AP conjugated (1:5000) Experiment design ① ② ③ Protein A ① Rabbit anti-human kappa 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Goat anti-human IgG (Fc specific)-AP conjugated (1:5000) Experiment design ① ② ③

16. Confirmation of antibody structure in cell cytosol using ELISA ΔLd kDa KV10 origin only HeLa cell ch3D8 IgG 50― 25― IB : Human IgG-Fc IB : Human IgG-kappa 10 ng 5 ng 1 ng Purrified ch3D8 IgG Goat anti-human IgG(Fc specific) #52 (1:3000) Goat anti-human Kappa chain #148 (1:3000) Rabbit anti-goat IgG(H+L)-HRP #56 (1:3000)

17. ① ② ③ Protein A ① ② ③ ① Goat anti-human IgG (Fc specific) 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Rabbit anti-human kappa (1:2000) ④ Goat anti-rabbit IgG (Fc specific)-AP conjugated (1:5000) ① ② ③ ④ ① Protein L coating 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Goat anti-human IgG (Fc specific)-AP conjugated (1:5000) ① ② ③ Protein L ① Protein A coating 1 μg/ml ② Cellulary expressed DNA cell lysate ③ F(ab’)2-Goat anti-human kappa-AP conjugated (1:5000) ① Rabbit anti-human kappa 1 μg/ml ② Cellulary expressed DNA cell lysate ③ Goat anti-human IgG (Fc specific)-AP conjugated (1:5000)

18. Confirmation of antibody structure in cell cytosol using IP ΔLd kDa KV10 origin only HeLa cell ch3D8 IgG 6C407 IgG 9C353 IgG 50― 25― IB : Human IgG-Fc IB : Human IgG-kappa Input Data REDUCING CONDITION HeLa cell

19. Confirmation of antibody structure in cell cytosol using IP IP : Human IgG-Fc specific REDUCING CONDITION HeLa cell Harvested after 24hrs KV10 origin IP : Human IgG-Fc IB : Human IgG-Fc IP : Human IgG-Fc IB : Human IgG-kappa only HeLa cell ch3D8 IgG 6C407 IgG 9C535 IgG ΔLd kDa 50― 25―

20. Confirmation of antibody structure in cell cytosol using IP ΔLd kDa KV10 origin only HeLa cell ch3D8 IgG 6C407 IgG 9C353 IgG 50― 25― IP : Human IgG-kappa IB : Human IgG-Fc IP : Human IgG-kappa IB : Human IgG-kappa IP : Human IgG kappa REDUCING CONDITION HeLa cell

21. Chimeric 3D8 IgG ①Cell penetration ② Nucleic acid binding Chimeric 3D8 IgG Murin Human Feature of chimeric 3D8 IgG

22. Confirmation of antibody structure in cell cytosol when purified protein is treated using confocal microscopy ① Goat anti-human IgG Fc specific ② Donkey anti-goat IgG (H+L)-Rodamine ③ Rabbit anti-human kappa chain ④ Mouse anti-rabbit IgG, IgM, IgA heavy chain-FITC HeLa cell seeding ↓ Chimeric 3D8 IgG : 5 μM (37°C,6h) ↓ Fixation, Permeabilization ↓ Goat anti-human IgG Fc specific (1:200) Rabbit anti-human kappa chain (1:200) (4°C, O/N) ↓ Donkey anti-goat IgG (H+L)-Rodamine (1:200) Mouse anti-rabbit IgG, IgM, IgA heavy chain-FITC (1:200) (RT, 1 hrs) FITC Roda mine ch3D8 IgG ① ② ③ ④

23. Plan Check interaction with 3D8 IgG and O2F3 antibody in reducing condition using ELISA Confirmation of antibody form in cell cytosol using –ELISA –Western blot –IP Confirmation of antibody form in cell cytosol when purified protein is treated using confocal microscopy