Streptactin xt beads

Glutathione S‐transferase‐fused SMS1‐CT and SMSr‐NT were bacterially expressed and highly purified using glutathione‐Sepharose beads (GE Healthcare). Twin‐Strep (TS)‐tagged DGKζ (TS‐DGKζ) was expressed by mammalian cells and highly purified using Strep‐Tactin XT beads (IBA Lifesciences, Goettingen, Germany).
Glutathione S‐transferase pull‐down assays were performed as previously [42 (link)]. Purified GST‐SMS1‐CT or SMSr‐NT were incubated with glutathione‐Sepharose beads for 30 min at 4 °C with constant rocking. The beads were washed five times with buffer containing 20 mm Tris–HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1% (v/v) Triton X‐100, and 1 mm phenylmethylsulfonyl fluoride. Purified Twin‐strep tagged DGKζ was incubated with the beads for 2 h at 4 °C with constant rocking. Then, the beads were washed five times with buffer. The washed beads washed were then boiled in SDS sample buffer, and the extracts were analyzed by western blotting.

Deletion fragments of p180 (short isoform NM_001042576, residues 29-381) and KTN1 (NM_001079521, residues 29–400) as well as full-length CLIMP63 were expressed as fusions with mNeonGreen-2×Strep in HEK 293T cells. 48 h post-transfection, cells were lysed in PBS (Lonza) plus 500 mM NaCl, 1% Triton X-100, and protease inhibitors and then centrifugated at 30,000g at 4 °C for 30 min. Supernatants were combined with Strep-Tactin XT beads (IBA Lifesciences) and rotated gently for 3 h. After extensive washing with lysis buffer (PBS plus 500 mM NaCl and 1% Triton X-100) and then wash buffer (IBA Lifesciences), bound proteins were eluted with Strep-Tactin XT Elution Buffer (IBA Lifesciences). Eluted proteins were subjected to multiple rounds of PBS dilution and concentration using 10 kDa protein concentrators (Sigma-Aldrich), before being aliquoted and frozen in liquid nitrogen.