After washing with ice-cold PBS, cells were either lysed in laemmli sample buffer or NP40 lysis buffer with a protease and phosphatase inhibitor cocktail (Roche).
Cell pellets used for detection of phosphorylated STAT3 were lysed in same buffer with additional phosphatase inhibitors (Roche).
After blue LED light exposure, the 661 W cells were lysed in a cell lysis buffer (RIPA buffer) with phosphatase inhibitor cocktails 2 and 3 (P5726 and P0044, Sigma-Aldrich) and a protease inhibitor (P8340, Sigma-Aldrich).
Slices were homogenized at 4 °C in an ice-cold lysis buffer with protease inhibitors, phosphatase inhibitors, 0.32 M sucrose, 1 mM HEPES, 0.1 mM PMSF, 1 mM MgCl2 using a glass-Teflon homogenizer (VWR, Radnor, PA, USA).
Tumor samples (at least three for each animal model and each treatment) were homogenized on ice in RIPA lysis buffer with protease and phosphatase inhibitors (Sigma-Aldrich) using a dounce homogenizer followed by passages through a 26-gauge needle.
Whole cell lysates were generated using RIPA buffer with phosphatase inhibitor and protease inhibitor cocktail at a concentration of 1×10 cells/μL and boiled for 10 minutes in protein sample buffer.
After the indicated treatments, cells were lysed in a Triton X-100 or RIPA buffer with phosphatase inhibitors.
Confluent monolayers of Caco-2 cells grown on solid plastic were treated for 24 h with 1 mM AICAR (with untreated cells as a control) before lysing in RIPA buffer with protease and phosphatase inhibitors (both at 1%).
Frozen tumor tissue was thawed on ice, immediately immersed in 500 μl RIPA buffer with proteinase and phosphatase inhibitors (Roche), and transferred to a Precellys Ceramic Kit tube (Peqlab).
For the western blot assay cell lysates of hMVEC were prepared by scraping the wells on ice using a rubber policeman and by adding Roche lysis buffer with phosphatase and protease inhibitors (Roche, Mannheim, Germany).
