Glutathione agarose beads

GST expression was induced by adding 1 mM isopropyl-β-D-thiogalactoside to bacteria containing pGEX-4T-1, and the protein was isolated from bacterial lysates by affinity chromatography with glutathione-agarose beads (Sigma-Aldrich, San Luis, MO, USA). For lysis, the bacteria were sonicated in NP40 buffer. Then, 6His fusion proteins were produced after the addition of 1 mM isopropyl-β-D-thiogalactoside to E. coli XL1-Blue containing derivatives of pQE30, purified in Ni-NTA agarose beads (Sigma-Aldrich), and eluted with 300 mM imidazole in binding buffer (50 mM NaH₂PO₄, 300 mM NaCl).

One mg of total proteins from C. elegans lysate were incubated on ice 10 min in 800 µL of TUBE lysis buffer [50 mM sodium fluoride, 5 mM tetra-sodium pyrophosphate, 10 mM β-glyceropyrophosphate, 1% Igepal CA-630, 2 mM EDTA, 20 mM Na₂HPO₄, 20 mM NaH₂PO₄, and 1.2 mg/ml complete protease inhibitor cocktail (Roche, Basel, Switzerland)] supplemented with 200 µg of purified LC3 traps or GST control (Quinet et al., 2022 (link)). After cold centrifugation at 16,200 g for 30 min, supernatant was harvested and added to 400 µl of prewashed glutathione-agarose beads (Sigma), and incubated for 6 hr rotating at 4 °C. Beads were centrifugated at 1000 g for 5 min at 4 °C (Beckman Coulter Microfuge 22 R, Fullerton, CA, USA), washed five times using 10 column volumes of PBS-tween 0.05%. Elution was done in 100 µL of (Tris pH7.5, 150 mM NaCl, 1% Triton, 1% SDS) at 95 °C during 10 min, and supernatant was harvested.

The initial ssDNA library and primers (HPLC-purified), Prime Time Gene Expression Master Mix and LNA probe for qPCR were obtained from Integrated DNA Technologies (Coralville, IA, USA). Graphene oxide, pGEX6P-1 vector, glutathione-agarose beads were purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptavidin-sepharose beads purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA) were used for separating biotinylated-ssDNA. SYBER gold nucleic acid gel stain was purchased from Thermo Scientific (Waltham, MA, USA). PCR reactions were performed using a PTC-100 Programmable Thermal Controller from MJ Research, Inc. (Waltham, MA, USA). qPCR reactions were run on a LightCycler 2.0 System (Roche Diagnostics Corporation, Indianapolis, IN, USA). Gels were visualized with a Gel Logic 112 Transilluminator, Carestream Health (Woodbridge, CT, USA) DNA concentrations were determined using a Thermo Scientific NanoDrop-2000 spectrophotometer.

The BL21 strain of Escherichia coli was transformed with plasmid pGEX‐STRA8 (full length and corresponding deletions), pGEX‐MYOD or with pGEX‐4T1 for expression of GST and grown at 37°C in LB medium until A₆₀₀ was 0.7, at which time isopropyl‐β‐thiogalactopyranoside (IPTG) was added for 3 hours at a final concentration of 0.5 mmol/L. Bacteria were collected by centrifugation and the pellet was resuspended in GST‐extraction buffer (GEB: 20 mmol/L Tris‐HC,l pH 7.4, 1 mol/L NaCl and 0.2 mmol/L EDTA) with a PIC (Sigma‐Aldrich). The suspensions were sonicated and debris removed by centrifugation. Fusion proteins were affinity purified by adsorption to glutathione‐agarose beads (Sigma‐Aldrich) for 2 hours at 4°C and eluted in the Elution Buffer (EB: 20 mmol/L Glutathione, 100 mmol/L Hepes pH 7.6, 1 mmol/L DTT and 0.1 mmol/L EDTA. Protein concentrations were determined by SDS‐polyacrylamide gel electrophoresis using purified BSA as standard and Coomassie gel staining.

E.coli BL21 transformed with plasmids encoding GST or GST fusion proteins were induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 h at room temperature. The cells were pelleted, washed once with PBS, and resuspended in PBS containing lysozyme and phenylmethylsulfonyl fluoride (PMSF). After sonicating twice, Triton X-100 was added to a final concentration of 1% and the mixture rotated at 4 °C for 1 h. After pelleting at 10,000× g for 10 min to remove cellular debris, the supernatant was incubated with glutathione agarose beads (Sigma-Aldrich Inc., St. Louis, MO, USA) at 4 °C overnight. After washing the beads 5 times with PBS, bound proteins were eluted using glutathione elution buffer (10 mM glutathione, 50 mM Tris-HCl, pH 8.5) and then dialyzed against PBS overnight. Concentrations were determined with a bicinchoninic acid protein (BCA) kit according to the manufacturer’s instructions (Pierce Biotechnology Inc., now part of Thermo Fisher Scientific, Waltham, MA, USA). The purity was assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining.