Mammalian cell lysis buffer

Serum starved monolayers of hRPE or ARPE19 cells (80–90% confluence) on 60 mm plates in 5% CO₂/95% air at 37 °C were challenged for 2 h with 0–40 μM HOHA lactone solutions (2 ml) in the respective basal cell culture medium. Cells were then scraped with a rubber policeman, transferred to 5-ml conical tubes and centrifuged at 480 g at 4 °C for 10 min. The medium was carefully aspirated and the cells were washed three times with ice-cold PBS followed by centrifugation at 480 g at 4 °C for 10 min. Cells were disrupted upon incubation with an ice-cold mammalian cell lysis buffer (GoldBio, St. Louis, MO) supplemented with 1x HALT protease inhibitor cocktail (Fisher Scientific, Pittsburgh, PA) for 15 min at 4 °C. Cell lysate was further placed on ice and sonicated at 40% power (5 cycles of 5 secs on and 5 secs off). The cell lysate was spun at 14,000 g at 4 °C for 20 min and the supernatant was collected and snap-frozen in liquid nitrogen. For PAGE analysis the supernatants were thawed out on ice and protein concentrations were determined using the 660 nm Pierce protein quantitation assay supplemented with ionic detergent compatibility reagent according to the manufacturer’s manual.