Protein g magnetic beads

Cardiac tissues were lysed in native lysis buffer (Solarbio Life Sciences) supplemented with complete protease inhibitor cocktail (Roche), PMSF and Na₂VO₃. The lysates were incubated with anti-O-GlcNAC (#ab2739, Abcam) antibody at 4 °C overnight. Antibody-bound proteins were precipitated with protein G magnetic beads (#1614023, Bio-Rad) and washed three times with lysis buffer. Enriched O-GlcNAc peptide samples were dissolved and loaded for LC separation on a NanoElute UHPLC system (Bruker Daltonics). Enriched peptides were analyzed on a timsTOF Pro mass spectrometer (Bruker Daltonics), equipped a ReproSil-Pur Basic C18 column. All mass spectrometric data were searched with Maxquant (v 1.6.1.0) and against Uniprot Mus_musculus_10090 database concatenated with reverse decoy database. Label free MS¹ based quantification of peptides was performed using MassChroQ. Retention time alignment between LC-MS data files was performed using the obiwarp method. The HA/Control ratio of each O-GlcNAc peptide was estimated by the average value of 3 hearts in each group, and the ratio >1.5 was define as hyperglycosylated. Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/).

QBC939 or RBE cells at 80-90% confluency in two 10-cm diameter dishes were scraped on ice into cold phosphate-buffered saline (PBS), pooled and lysed with 1 ml of Pierce™ IP lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with protease inhibitors (Roche Diagnostics). Protein G magnetic beads (Bio-Rad Laboratories, Inc.) were washed three times with PBS-0.1% Tween-20 and incubated with 3 µg of antibody in a final volume of 200 µl for 30 min at room temperature. The beads were washed three times and incubated overnight at 4°C. The beads were washed again, and 30 µl of 1X loading buffer was added. The slurry was incubated at 100°C for 10 min, and the beads were discarded. The supernatant was collected for western blotting conducted as described below. The following antibodies were used: GATA6 (cat. no. 5851; Cell Signaling Technology, Inc.); LOXL2 (cat. no. MAB2639; R&D Systems, Inc.); Flag (cat. no. 14793; Cell Signaling Technology, Inc.); rabbit IgG (cat. no. bs-0295P; BIOSS), and mouse IgG (cat. no. bs-0296P; BIOSS). HRP-conjugated VeriBlot for IP (1:1,000; cat. no. ab131366; Abcam) was used as the secondary antibody, and the membrane was incubated for 2 h at room temperature.

Briefly, rG4IP was performed with a structure-specific G quadruplex (BG4) antibody (Absolute Antibody). TCC-S cells (15 × 10⁶) were collected, washed with ice-cold PBS, and resuspended in ice-cold lysis buffer (150 mM KCL, 50 mM HEPES, 25 μg/mL Digitonin, 100 U/mL RNase inhibitor). The cells were incubated with lysis buffer for 10 min at 4°C using end over end rotation and centrifuged at 2,000 × g for 5 min at 4°C. The supernatant (cytosolic fraction) was saved and 10% was removed to be used as input control. When transfections were required, 1 × 10⁶ DU145 cells were seeded in a 100 mm dish, transfected using JetPRIME transfection reagent (Polyplus), trypsinised after 48 h, and processed as above. Precleared lysates were incubated overnight with 3 μg of BG4 antibody bound to Protein G magnetic beads (Biorad). After incubation, the beads were magnetised, washed thrice with lysis buffer, and eluted by incubating at 65°C for 15 min to release the bound nucleic acids. The eluent was treated with 2 U of RNase-free DNase I (ThermoFisher) for 15 min at 37°C to remove contaminating DNA. The RNAs from input and IP fractions were then isolated through TRIzol (ThermoFisher) extraction followed by isopropanol precipitation.

4-day-old cry2-1;UBQ10_pro:UBP13-6xHA;CRY2_pro:2xStrep-6xHis-3xFLAG-CRY2 seedlings grown under continuous white light were dark adapted for 24 h, then treated with continued darkness or UVC LED source (approximately 1800 J/m²). Tissue was collected after 10 min, immediately frozen and later ground in liquid nitrogen. Each 1 g of tissue was dissolved in 2 ml of SII buffer (100 mM sodium phosphate [pH 8], 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Triton X-100, 1× protease inhibitors (Sigma), 50 μM MG132) and sonicated (Branson Ultrasonics) on the ice at 40% power, with 0.5 s on/off cycles for a total of 10 s. The protein extracts were then clarified by two rounds of centrifugation at 13000 rpm for 10 min at 4°C. Protein concentration was inferred by spectroscopy using Bradford reagent (Bio-Rad), and normalized for inputs and co-IPs. For co-IPs, proteins were then mixed with anti- FLAG antibody (Thermal Fisher Scientific) for 1 h at 4 °C and incubated with protein-G magnetic beads (Bio-rad) for 0.5 h at 4 °C. Beads were washed 3× with 0.75 ml of SII buffer and proteins were eluted with 20 μl of 2× LDS buffer and boiled at 95°C for 5 min before immunoblot analysis, as described above.