Anti his primary antibody

Microsomal proteins were isolated from the clone five transformed Pichia cells as described in Basu et al. [17 (link)]. For immunoblot analysis, 5 μg of microsomal protein from Pichia transformants was denatured, subjected to 10% SDS-PAGE, and electroblotted onto PVDF Immobilon membranes (Millipore) using the Mini Protean3 system according to manufacturer's recommendations. Blots were probed with an anti-His primary antibody (Clontech) at a 1:10,000 dilution and a secondary goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) (Clontech) at a 1:20,000 dilution. West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used for HRP detection. Pichia cell lines transformed with the empty expression vector (pPCIZ B) were used as the negative control (NC). Protein quantification was done using the Bradford reagent (Sigma). Blots were stained with Coomassie Brilliant Blue R-250 (Sigma) following HRP detection to ensure equal loading.

INpro expression in E. coli cells and the presence of purified INpro in fractions from chromatographic methods was confirmed by SDS-PAGE analysis (e.g., Supplementary Figure S3). Western blot analysis was performed using a primary anti-INpro-1,205 rabbit polyclonal antibody, which targets the residues 1,205–1,220 (CMAGDQSRLTAGKNS) custom-made by Genscript. The blots were probed with a goat anti rabbit secondary antibody (Rockland, United States; 1:5000). Also, Western blots using Anti-His primary antibody (Clontech; 1:4000 dilution) was performed with an anti-mouse HRP conjugate secondary antibody (Dako; 1,3,000) to validate the presence of the protein construct.