μ columns

Nycodenz cell fractionation methods were performed as previously described (Fox et al., 2020 (link)). Briefly, mice were deeply anesthetized with isoflurane and decapitated. Brains were removed, mechanically disrupted, and fractionated using a discontinuous Nycodenz gradient (Stromhaug et al., 1998 (link)). Isolated fractions were then examined by immunoblotting or further purified for LC3+ structures by immuno-isolation (Jeong et al., 2009 (link)). For immuno-isolation, 30 μg of the AV fraction were incubated with 4 μg of rabbit anti-LC3A/B (Abcam) in 1x sucrose-HEPES buffer with Halt protease inhibitor cocktail (ThermoFisher) and without detergent overnight at 4°C. Subsequent immune-vesicle structure complexes were incubated with Protein A MicroBeads (Miltenyi Biotec) for 1 h at 4°C and isolated using μ columns (Miltenyi Biotec).

Proteins from 5-week-old SnRK1α1–GFP, SnRK1α1ΔKA1–GFP, SnRK1α1–GFP_siz1-2 or 35S::GFP plant leaves were extracted with immunoprecipitation (IP) buffer [50 mM Tris–HCl pH 8.0, 50 mm NaCl, 1% (V/V) Igepal CA-630, 0.5% (w/V) sodium deoxycholate, 0.1% (w/V) SDS, 1 mM EDTA pH 8.0, 50 μM MG132, 50 mM N-ethylmaleimide and cOmplete protease inhibitor cocktail (one tablet/10 mL)]. After clearing samples by centrifugation (6785 g, 2°C, 10 min) 800 μL of supernatant were supplemented with fresh MG132 (50 μm) and incubated at 4°C for 1 h with 40 μL of μMACS anti–GFP MicroBeads (μMACS GFP Isolation Kit, Miltenyi, 130-091-125). Samples were thereafter loaded in μColumns (Miltenyi Biotec, Bergisch Gladbach, Germany 130-042-701) pre-equilibrated with 1 mL of IP buffer, and allowed to flow through. Columns were washed three times with 200 μL and once with 600 μL of IP buffer and proteins eluted with 80 μL of elution buffer (Miltenyi, 130-091-125) at 95°C. β-Mercaptoethanol (2%) was added to the eluates prior to boiling for 5 min at 95°C. Proteins were resolved by SDS-PAGE, wet-transferred to a PVDF membrane (30 V, 16 h at 4°C), and analysed by immunoblotting with SnRK1α1, SnRK1β1, SnRK1γ, SnRK1βγ, SUMO1, UBQ11 and GFP antibodies. For each GFP immunoprecipitation experiment, immunodetection with different antibodies was done using equal loading on independent membranes.