Additional file 4 of An antibody-drug conjugate targeting soluble and membrane-bound TGFα is effective against pancreatic tumors

Supplementary Material 4: Fig. 4. A. Stain-free gels (top) were used to evaluate the binding of anti-TGFα antibodies to the cytotoxic agents DM1, MMAF, and DXd. A change in the migration of the antibody in the gel was observed after coupling. Bottom blots: one hundred nanograms of each ADC and the unconjugated mAbs were loaded in 12% SDS-PAGE gels. The cytotoxic payload bound to the light and heavy chains of the ADCs was analyzed by Western blot using anti-DM1, anti-MMAF, and anti-DXd antibodies, as shown below. B. Detection of DM1 bound to 5F1 or 16B10 mAbs, compared to T-DM1. Equal amounts of the nude or coupled antibodies were used. Blots were analyzed using anti-DM1 antibodies. C. Restoration of 16B10-MMAF sensitivity in a TGFA-KO clone. The parental cell line IMIM-PC2, the CRISPR/Cas9 clone #25 and the TGFA reconstituted clone #13 were seeded in 6-well plates and treated with 2.5 nM of the ADCs (colored boxes). After 5 days, the effect on cell proliferation was assessed by cell counting. The results obtained are represented as the mean ± SD of the triplicates of an experiment that were repeated three times. **P < 0.01, calculated by Mann-Whitney U test.