SDS-PAGE and Western blots

For protein expression and inteins splicing evaluation, samples in 1× Laemmli buffer were boiled for 10 min and spun down for 10 min at 17,000 × g. Routinely, 10–15 μL of sample was separated in Any kD TGX Stain-Free protein gels (4568126, Bio-Rad) or 4–20% TGX stain-free protein gels (4568095, Bio-Rad). Proteins were stained with Bio-Safe Coomassie Stain (1610786, Bio-Rad) or transferred to nitrocellulose membranes (1704270, Bio-Rad) using the Trans-Blot Turbo Transfer System (1704150, Bio-Rad), according to the manufacturer’s instructions. Precision Plus Protein Dual Xtra Prestained Protein Standards (1610377, Bio-Rad) and Chameleon Duo Prestained Protein Ladder (928-60000, Li-cor) were used for Coomassie stained gels or Western blots, respectively. Western blots and near-infrared detection were performed as described in Li-cor “Near-Infrared Western Blot Detection document” (Doc. #988-13627), using 5% (w/v) skimmed milk in 1× tris-buffered saline (1706435, Bio-Rad) for blocking. Antibodies recognizing residues 27–41 of the mCherry (Anti RFP-tag, pAb, Rabbit; A00682, GenScript) or the hexahistidine tag (THE His Tag Antibody, mAb, Mouse; A00186, GenScript) were used as primary antibodies at dilutions of 1:3000 and 1:5000, respectively. IRDye 680RD Goat anti-Rabbit (925-68071, Li-cor) and IRDye 800CW Goat anti-Mouse (925-32210, Li-cor) were used as secondary antibodies at dilutions of 1:20,000. Stain-free images of TGX stain-free protein gels were acquired and analyzed using Image Lab v5.2 (Bio-Rad). Coomassie stained protein gels and blotted membranes were visualized using an Odyssey CLx Infrared Imaging System (Li-cor) and analyzed using the Image Studio Lite v5.2.5 software (Li-cor). The theoretical molecular weight for all proteins analyzed by SDS-PAGE or Western blot are described in Supplementary Table ⁹.