Anti c myc agarose beads

Expression vectors encoding HA-MKK7, flag-septin 11-WT (wild-type), and c-Myc-GADD45β (GenScript Inc.) driven by CMV were transfected into HEK-293FT cells. To test the requirement for septin 11-MKK7 interaction in the role of septin 11 in cellular responses to 27HC, flag-septin 11-∆G4 and flag-septin 11-∆GTP were also generated. All constructs were confirmed by sequencing. Seventy-two hours post-transfection, the cells were pelleted and lysed, and proteins were extracted using a buffer containing 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, and protease inhibitors. The recombinant proteins were purified using anti-HA agarose (Thermo Fisher Scientific, 26181), anti-flag M2 affinity gel (Sigma, A2220), or anti-c-Myc agarose beads (ThermoFisher Scientific, 20168), and elution with 0.1 M glycine, pH3.5, followed by neutralization to pH 7.0. Proteins were freshly prepared prior to use. In pull-down assays the beads were pre-blocked with 1% BSA to reduce nonspecific binding, and the bait protein was added and incubated with beads for 1 h at 37 °C. After extensive washing the prey protein(s) was added and incubation resumed at 37 °C for 1 h. Following washing X3, protein complexes were eluted and samples were subjected to SDS-PAGE and immunoblot analysis.

HEK293A cells were transfected with pcDNA3.0-BMAL1-His, PcDNA3.0-6XMyc-CLOCK or PcDNA3.0-6XMyc-CLOCKC267A using Polyethylenimine PEI MAX reagent (Polysciences 247,651). Twenty-four hours after transient transfection, cells were treated with the indicated compounds for 8 h and homogenized in lysis buffer (50 mM Tris pH7.5, 150 mM NaCl 0.5% Triton X-100, 5% glycerol and protease inhibitor). Immunoprecipitation was performed overnight using Anti-c-Myc Agarose beads (Thermo Fisher 20168). Beads were washed three times with lysis buffer. The resultant protein samples were resolved by SDS-PAGE and immunoblotted with antibodies that are described in Supplemental Table 3.