Tgx stain free protein gel

The constructs encoding for GST-fused LC3B, GABARAP, and their respective mutants were expressed in E. coli BL21 (DE3) cells, and the expression of the fusion proteins was induced by addition of 0.5 mM IPTG at 37  °C over-night. Cells were lysed by sonication in lysis buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 5 mM EGTA, 150 mM NaCl) and Glutathione Sepharose 4B beads (GE Healthcare) were used to precipitate the GST-fused proteins. Fusion protein-bound beads (5 μl and 15 μl for the GABARAP and LC3B pull-downs, respectively) were used directly in GST pull-down assays. HEK293 (400 μg and 800 μg of total proteins for the GABARAP and LC3B pull-downs, respectively) were lysed in lysis buffer (50 mM TRIS, pH 7.5, 150 mM NaCI, 1 mM EDTA, 0.5% Triton X-100, supplemented with protease and phosphatase inhibitors). Lysates were cleared by centrifugation at 16,000 × g for 10 min, and incubated with GST fusion protein-loaded beads for 2 h, at 4 °C. Beads were then washed four times in lysis buffer, resuspended in NuPAGE® LDS Sample Buffer (Life Technologies) supplemented with 1% β-Mercaptoethanol and boiled. Supernatants were loaded on 4–15% TGX Stain-Free protein gels (Biorad) for SDS-PAGE and the immunoblot was performed as described above. GST and GST-fused proteins were visualized by gel activation (Stain-Free Imaging Technology, Biorad), as a substitute of the Coomassie Blue staining.

Mice were sacrificed by CO₂ inhalation, and the vasculature was perfused with phosphate-buffered saline, via the left ventricle, to remove all blood. The aortas from E19, P15, and P90 were then carefully excised (free of fat) from the root down to the diaphragm. Vessels were snap frozen in liquid nitrogen and stored at −80 °C. For Western blot analysis, the frozen aortas were then homogenized using the Bead Ruptor 4 Homogenizer (Omni International) in 200 µL of RIPA buffer (Milliporesigma, R0278, Burlington, MA, USA) containing complete EDTA-free Protease Inhibitor Cocktail (MilliporeSigma, 11873580001, Burlington, MA, USA), and protein was quantified with Pierce Rapid Gold BCA Protein Assay kit (Thermo Scientific, A53226). A total of 30 µg of total protein was loaded on TGX stain-free protein gels (Bio-Rad, 17000927, Hercules, CA, USA) for blotting.

Plasmids pCHAP7802 encoding wild-type PulF or its cysteine-substituted derivatives were transformed into the E. coli PAP7460 strain. Bacteria were grown overnight in LB in the presence of ampicillin at 100 µg mL⁻¹ at 30°C. The next day, 200 µL of the overnight culture was inoculated into 5 mL of fresh media and incubated at 30°C to an OD_600nm of ~2. One OD_600nm of bacteria was harvested by centrifugation (12,000 × g for 5 min) and washed twice with phosphate buffer saline (PBS). Bacteria were resuspended in 1 mL of the cross-linking buffer (50 mM MOPS pH 7.0, 5 mM MgCl₂, 10% glycerol) and incubated 10 min at 23°C in a thermomixer with shaking at 750 rpm. For oxidation, 30 μL of 10 mM CuCl₂ was added to each sample. After an incubation of 23 min at 23°C, the reaction was stopped by adding 45 μL of 0.5 M EDTA. Bacteria were centrifuged and resuspended in 100 µL of the SDS sample buffer (87 (link)). Samples were boiled for 10 min and 20 μL was analyzed by 10% glycine, or 8% tricine SDS-PAGE, or using 4%–15% TGX stain-free protein gels (Biorad). Proteins were transferred on nitrocellulose membranes and probed with anti-PulF_N or anti-PulF_C antibodies. Each variant was checked for cross-linking three times, and the variants which showed a reproducible cross-linked band were considered as positive.