After incubation for 24 h, cells were washed once with 2 ml of ice-cold PBS for 10 minutes, extracted three times with 2 ml of cold 10% TCA for 5 minutes each time, and solubilized for at least 30 minutes at room temperature in 0.2 ml 0.3 N NaOH, 1% SDS.
The lysosome fraction was collected, washed three times in PBS (18,000 g for 30 minutes at 4°C), and solubilized in an extraction buffer containing 10 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 0.2% SDS, and protease inhibitors (as above).
The feces were weighed and then dissolved in 4 mL of PBS by vigorous shaking, while the urine samples were spin for 10 minutes at 11000 g and solubilized in 100 μL.
Briefly, cells were incubated in TKM buffer (50 mM Tris-HCl pH 7.4, 25 mM KCl, 5 mM MgCl2 and 1 mM EGTA) containing 1% Triton and protease inhibitor cocktail CLAP [10 µg/ml chymostatin, leupeptin, antipain and pepstatin A (Sigma) for 30 minutes on ice, sonicated for 2 minutes and centrifuged at 5000 g for 30 minutes. The pellet and the supernatant were separated and solubilized in sample buffer.
The centrifugation pellet was resuspended in 1 ml HEPES-EDTA buffer and solubilized in 0.6% v/v SurfactAmps Brij58 (Pierce Endogen, Rockford, IL, USA) for 30 minutes at room temperature.
DOC-insoluble material was isolated by centrifugation and solubilized afterwards in 40 μl sodium dodecylsulfate (SDS) buffer.
It was collected, lyophilized, and solubilized in deuterium oxide.
We then took the pellet fraction and solubilized it by the addition of 8 M urea.
Finally, precipitated proteins were washed and solubilized in Tris buffer 10 mM.
The unbound fraction was collected and solubilized in 2XSB.
