Glutathione agarose beads

Polyclonal rabbit anti-human CCDC88B antiserum was prepared as we have previously described^{5 (link),34 (link)}. A recombinant protein consisting of glutathione-S-transferase (GST) fused in-frame to a CCDC88B segment corresponding to human amino acid positions 650 to 769, was expressed in E. coli BL21, followed by affinity purification with glutathione-agarose beads (GE Healthcare). Antisera were raised in New Zealand white rabbits using purified protein (0.5 mg per rabbit per injection) emulsified in Freund’s incomplete adjuvant. Affinity purification of the anti-CCDC88B antibody^{35 (link)} was using a recombinant CCDC88B protein (positions 650 to 769) comprising a poly-histidine tail (His)6 fused in-frame at its N terminus, and purified by chromatography onto Ni-NTA agarose (Qiagen). The immobilized protein was used to capture the anti-CCDC88B fraction of the hyperimmune serum, which was then released by washing with imidazole-containing buffer^{34 (link)}. The specificity of the anti-CCDC88B antibody was tested by western blotting total cell extracts from HEK293T control cells and HEK293T cells (ATCC, CRL-11268) stably expressing a full-length human CCDC88B.

The ORF of SiMYB3 was fused in-frame with GST in p4T-1 and expressed in E. coli DE3, and purified by standard procedures using glutathione agarose beads (GE Healthcare, Pittsburgh, PA, USA). Briefly, 5 mL of DE3 cells grown overnight and expressing the desired constructs were transferred into 500 mL of LB and grown at 37 °C for 3 h (OD = 1.0). Isopropyl-β-d-thiogalactopyranoside (IPTG, 1 mM) was then added to the media and incubated overnight at 16 °C to induce protein expression. The bacterial cells were sonicated in PBS with 1% Triton X-100 and centrifuged at 10,000 g for 10 min to remove insoluble cell debris. The supernatant was incubated with PBS pre-equilibrated with lutathione agarose beads and rotated at 4 °C for 4 h. After washing five times with PBS, GST-tagged protein was eluted using 10 mM glutathione. For EMSA, 30 ng of purified GST-SiMYB3 recombinant protein, 400 fmol of biotin-labeled annealed oligonucleotides, 2 μL of 10× binding buffer (100 mM Tris, 500 mM KCL, and 10 mM DTT, pH 7.5), 1 μL of 50% (v/v) glycerol, 1 μL of 100 mM MgCl2, 1 μL of 1 μg/μL poly (dI-dC), and 1 μL of 1% (v/v) NP–40 were combined and double-distilled water was added to a final volume of 20 μL. Biotin-labeled DNA was detected using the LightShift Chemiluminescent EMSA kit (Thermo Scientific, 20148, Waltham, MA, USA).

The full-length CaM1 was constructed into the pGEX4T-1 vector, while the cDNA for CNGC12 (CT) was subcloned into the pET28a vector to generate the pET28a-CNGC12-CT fusion construct. The vectors were expressed in the Escherichia coli strain Rosetta DE3 (Stratagene). When cells were grown to an OD₆₀₀ of 0.6, they were induced with 1 mM isopropyl-β-d-thiogalactoside (IPTG) for 5 h at 37°C. For purification of the glutathione S-transferase (GST)-tagged fusion proteins, the cells were lysed by sonication on ice in phosphate-buffered saline (PBS) buffer containing protease inhibitor cocktail (Roche), 1 mM lysozyme, and 1% Triton X-100. The pellets and supernatants were separately collected by centrifugation at 12,000 g for 20 min at 4°C. The pellets were washed with the PBS solution four times. For GST fusion protein purification, the supernatants were purified using glutathione agarose beads (GE) and subjected to western blot with anti-GST antibody.

Bacteriologically expressed GST fusion proteins were coded by pGEX plasmids. We used BL21 strains of Escherichia coli for amplification of the pGEX GST-NFAT plasmid and protein extraction. The transformed colonies were inoculated with 5 mL of LB medium (Roth) and 5 µL of ampicillin (Sigma-Aldrich), and the culture was incubated at 37 °C on an orbital shaker for 12–15 h (up to OD660 of 0.2–2.0). Expression of NFAT fusion proteins was induced by adding 0.75 mL of IPTG solution (AppliChem). Bacteria were lysed by sonification, and we identified the produced proteins by means of SDS–polyacrylamide gel electrophoresis. For the actual assay, we incubated 100 µL of purified glutathione agarose beads (GE Healthcare) with 3 µg of bacteriologically expressed GST or GST-NFAT and total protein at 4 °C for 15–18 h. After centrifugation and several wash cycles, samples were mixed with 30 µL of Laemmli puffer, heated up to 95 °C for 5 min, and analyzed by Western blotting.

GST‐SLC27A5 was purified by glutathione agarose beads (GE Healthcare, Piscataway, NJ, USA). His‐IGF2BP3 was purified by HisPur Ni‐NTAmagneticbeads (Thermo Fisher, MA, USA). The purified proteins were incubated with glutathione agarose beads for 3 h. The beads were washed with wash buffer (137 mm NaCl, 2.7 mm KCl, 10 mm Na₂HPO₄, 2 mm KH₂PO₄ and 0.5% Triton X‐100), mixed with 2× SDS loading buffer and boiled for 8 min. The inputs/elutions were further analyzed by Coomassie staining and/or immunoblot analysis.