For total protein extraction, mycelia grown in liquid CM and MM-N medium (used for nitrogen starvation, 0.5 g/L KCl, 0.5 g/L MgSO4, 1.5 g/L KH2PO4, 10 g/L glucose, pH 6.5) were ground into a fine powder in liquid nitrogen and resuspended in 0.6 mL extraction buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 2 mM PMSF and 1% Triton X-100). The supernatants were centrifuged 15,000 g for 25 min at 4°C to remove cell debris. Total protein concentration was measured using the Bio-Rad Protein Assay and separated on a 12.5% SDS PAGE gel and transferred to PVDF membranes for Western blot analysis. The expression of RFP-Atg8 was detected with anti-RFP (Clontech, USA). The horseradish peroxidase–conjugated secondary antibody and the ECL kit (Amersham Biosciences, Germany) were used to detect the chemiluminescent signals.
For the immunoprecipitation of GFP-fusion-proteins from cellular extracts, equal concentration of total proteins were isolated and incubated with 20–30 μL of GFP-Trap agarose beads (ChromoTek, Germany) and carried out as manufacturer’s instructions. Proteins eluted from GFP-Trap agarose beads were analyzed by immunoblot detection with the anti-RFP (Clontech, USA), anti-GFP (Sigma-Aldrich) antibodies and anti-Actin (Sigma-Aldrich).
For the immunoprecipitation of GFP-fusion-proteins from cellular extracts, equal concentration of total proteins were isolated and incubated with 20–30 μL of GFP-Trap agarose beads (ChromoTek, Germany) and carried out as manufacturer’s instructions. Proteins eluted from GFP-Trap agarose beads were analyzed by immunoblot detection with the anti-RFP (Clontech, USA), anti-GFP (Sigma-Aldrich) antibodies and anti-Actin (Sigma-Aldrich).