Sepharose fast flow beads

Cells were collected and lysed in 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1% Nonidet NP-40 substitute, supplemented with protease inhibitors, incubated for 15 min on ice; the nuclei were then pelleted at 1000 g for 15 min. Soluble material was quantified and 10 mg protein lysate incubated for 2 pre-clearing cycles with 30 µl protein A Sepharose fast flow beads (GE Healthcare, 17–0618–01) for 3 h. Pre-cleared lysate was incubated for 16 h with 30 µg beads preconjugated with 5 µg rabbit anti-SQSTM1 antiserum (Sigma-Aldrich, PA0067) or control serum. Beads were washed 4 times in 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 0.1% Nonidet NP-40 substitute, and resuspended in Laemmli buffer for SDS-PAGE resolution.

Recombinant p38α and constitutively active MKK6 (S207E/T211E) were purified as previously described (Mittelstadt et al., 2009 (link)). In brief, p38α and MKK6 were expressed in the bacterial strain BL21(DE3) using the vector pET15b. After cultures reached an A600 of 0.5–1.0, protein expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) and incubated overnight at 4°C. Cells were resuspended in ice-cold PBS, including 0.5 M NaCl, 0.1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, sonicated, and centrifuged at 20,000 g for 20 min at 4°C. Proteins were purified with cobalt-charged chelating–Sepharose Fast Flow beads (GE Healthcare) and eluted with 0.3 M imidazole in PBS with 0.5 M NaCl. Proteins were concentrated and washed into PBS using Microcon YM-30 spin columns (Millipore).