Total protein was extracted using radioimmunoprecipitation (RIPA) lysis buffer completed with phenylmethanesulfonyl fluoride (both from Beyotime Biotechnology).
The bilayer translation reaction mixture was incubated with 10 μl of MagneHis Protein Purification System (Promega) particles at 4°C for 30 minutes in 250 μl translation buffer completed with NaCl to 500 mM final concentration.
For pull-down, one brain of an adult P25-P300) mouse (ca. 0.4g) was homogenized in 20 ml using a glass-homogenizer in pull-down buffer completed with protease inhibitors (Roche), 1% Triton X-100, and 2 mM CaCl2.
Mouse macrophage cell line J774A.1 was cultured in DMEM/F12 containing 10% fetal bovine serum supplemented with 1% glutamine, penicillin and streptomycin (100 U/mL and 100 μg/mL respectively) and 10 mM Hepes buffer (complete medium).
Directly before addition of the lysis buffer, Complete protease inhibitors (Roche) and 1 mM PMSF were added.
Heads from age and sex-matched adults were disrupted in lysis buffer, complete protease inhibitor cocktail (Roche) using a sonicator-3000 froMISONIXIX.
The samples were homogenized using a hand-held homogenizer (Omni Intl., Warrenton, VA) on ice in 2 ml of protease inhibitor buffer (Complete Mini Protease Inhibitor Cocktail tablets at a proportion of 1 tablet/25 ml PBS, Roche Diagnostics, Indianapolis, IN).
Dissected mouse retinas were placed into 200 μl of protein lysate buffer (Complete Ultra, Roche) with proteinase inhibitor cocktail (Calbiochem, Gibbstown, NJ) at 4°C.
Briefly, frozen leaf tissues (1 g fresh weight) were ground in ice-cold buffer 100 mM, pH 8.0, Tris buffer (50 mL) containing 1 mM ethylenediamine tetraacetic acid, 1 mM dithiothreitol and a protease inhibitor cocktail (1 tablet for 100 mL of buffer, Complete Mini, Roche Diagnostics, Meylan, France).
A 5.0 ml aliquots of these solutions were transferred to 50 ml standard flask, the pH was adjusted to 7.0 using tris buffer and completed to the mark with water.
Then, the IPG strips were applied to a 7 cm IPG strip holder containing 60 μL of rehydration buffer to complete IEF for an additional 8 000−12 000 voltage hours, called refocusing IEF.
