This Data Analysis Problem does not appear in the textbook.

Source:  Koo, M. Y., J. Park, J. M. Lim, S. Y. Joo, S.-P. Shin, H. B. Shim, J. Chung, D. Kang, H. A. Woo, S. G. Rhee. 2014. Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody. Proc. Nat. Acad. Sci. USA 111: 6269–6274.

Corresponding chapter(s) in the textbook: Chapter 17 

Review the following terms before working on the problem: antibodies, immunoglobulins, variable fragments (VH and VL), expression vector, polypeptide chain, gene therapy, tyrosine-phosphorylation, signal transducers and activators of transcription (STATs), adenovirus vector, green fluorescent protein (GFP), hepatoma cells, cell lysate, Western blotting, actin, interleukins, cytokines, immunofluorescent staining, confocal microscopy

Experiment

Intrabodies are single-chain antibodies engineered by sophisticated recombinant DNA technology. They contain the variable fragments (VH and VL) of a specific immunoglobulin molecule held together by a short linker peptide. They can be expressed in the cell from an expression vector as a single polypeptide chain and inhibit the function of a target protein inside the cell. For this reason, they hold promise for the treatment of certain human diseases as gene therapy agents.

A single-chain antibody (designated scFv17) directed against the tyrosine-phosphorylated form of signal transducer and activator of transcription 3 (STAT3) protein was tested in this experiment (this protein is designated pYSTAT3). A recombinant adenovirus vector containing the DNA region coding for the intrabody against pYSTAT3, fused with the gene for green fluorescent protein (GFP), was constructed (A). An adenovirus coding for GFP only was used as a control. The two viruses are referred to as “GFP-scFv17” and “GFP,” respectively, in Figure B.

Hepatoma cells were infected with control GFP-viruses (samples 1 to 3) or GFP-scFv17-viruses (samples 4 to 6) at various particle:cell ratios (MOI, multiplicity of infection). Cell lysates were isolated, and Western blot analysis was performed using anti-pYSTAT3, anti-STAT3, anti-GFP, and anti-β-actin antibodies.

In another experiment (C) GFP- and GFP-scFv17-expressing cells were left untreated or were treated with interleukin-6 (IL-6), a cytokine. Immunofluorescent staining was performed using an anti-STAT3 antibody, and the same cells also were stained with diamidino-phenylindole (DAPI), a DNA-binding dye. The third column shows the merged fluorescence microscopic images of the two stainings.

Figure

Source: Koo, M. Y., J. Park, J. M. Lim, S. Y. Joo, S.-P. Shin, H. B. Shim, J. Chung, D. Kang, H. A. Woo, S. G. Rhee. 2014. Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody. Proc. Nat. Acad. Sci. USA 111: 6269–6274.

Questions

1. Why was anti-β-actin antibody used in the Western blot?

2. Why was anti-GFP antibody used in the Western blot?

3. What proteins correspond to bands a and b in Figure B?

4. How did the intracellular antibody affect STAT3 expression (B)?

5. How did the intracellular antibody affect the Tyr-phosphorylation of STAT3?

6. How does IL-6 affect STAT3 in the control cells (C)?

7. What was the effect of the single-chain antibody scFv17 on this process? Suggest an explanation.