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. 2019 Jul 3;10:744. doi: 10.3389/fphar.2019.00744

Figure 4.

Figure 4

Cell-free co-expression for production of ERBB (erythroblastic oncogene B) tyrosine kinase complexes. (A) Illustrated ERBB2 receptor embedded in an NLP via cell-free expression and self-assembly. (B) Analysis of the co-expression of EGFR (epidermal growth factor receptor) and ERBB2 in the presence of the plasmid encoding an ApoA1 gene via 4–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Visualization was accomplished via the incorporation of a fluorescent bodipy lysine dye. (C) NLP associated ERBB2 is tyrosine phosphorylated. Cell-free expressions were set up with and without (−) ERBB2 plasmid. Samples were collected at 2 h, and 5 h, resolved by SDS-PAGE and western blotted with anti-phospho-tyrosine ERBB2 antibody pY1248 and anti-ERBB2 antibody Ab-3 after stripping. (D) NLP associated EGFR is phosphorylated, and the presence of EGF in the cell-free reaction increases the level of phosphorylation. EGFR-NLPs showed low level of phosphorylation during cell-free expression. Adding EGF, the natural ligand of EGFR, increased the phosphorylation. Cell-free mixtures were resolved by SDS-PAGE and western blotted with anti-phospho-tyrosine EGFR antibody pY1110 and anti-EGFR. Images were spliced lanes from the same Western blot.