Fig. 7: Intra-domain disulfide bond is not detrimental functionally to the evolved VH-S4 domain and is retained in a reducing environment.

a 293FT cells were transfected for 48 h with FLAG-tagged empty vector (Mock), VH-S4(S22C-T92C) and VH-S4. Cell lysates were then subjected to Flag immunoprecipitation (IP) followed by western blot analysis. to detect the presence of endogenous eIF4E as co-immunoprecipitant in the IP. b Cells transfected as in a were cultured in presence or absence of MG132 for 18 h. Lysates were analysed by western blot and level of endogenous eIF4E and overexpressed Flag-tagged miniproteins were assessed using an anti-eIF4E and anti-Flag antibody. c Western blot analysis of whole cell lysates from 293FT cells transfected for 48 h with FLAG-tagged directed evolution VH mutants, mock and corresponding controls harbouring relevant amino acid replacements at position 22 and 92 as denoted. Removal of the W⁹⁵GGDGFYP^100B loop with SAAA is indicated. Cellular expression of VH domain proteins was assessed with anti-flag antibody over a short (S.E.) and long western blot exposure (L.E.) β-actin was used as loading control for all the experiment. d Recombinant VH-S4(S22C-T92C) and VH-S4 were analysed by liquid chromatography/mass spectrometry, after treatment with the indicated conditions. Mass spectra were integrated across the principle components of the resulting total ion chromatograms, and the experimental masses were taken as the average across individual charge state ions. The deconvoluted masses have experimental errors of ~300 ppm (based on molecular weights of 17,416.15 Da and 17,434.25 Da for VH-S4 and the reduced form of VH-S4(S22C-T92C), respectively), which are of the order expected for a single-quadrupole mass analyser. All western blot analyses were repeated twice to verify their reproducibility. Representative blots are shown. Source data for a and b are provided in the Source Data file.