Transfected NIH3T3 cells were grown for two days, and then starved overnight, PBS-washed and lysed with RIPA buffer and the lysate cleared by centrifugation at 10,000 g (10 min at 4°C).
In brief, cells were lysed using OpGen lysis buffer and the lysate diluted for direct use.
For total cellular protein, cells were lysed directly in RIPA buffer and the lysate was collected by centrifugation.
Cells were lysed for 30 minutes on ice in modified CHAPS buffer, and the lysate was centrifuged at 115,000 ×g, at 4°C for 1 hour.
For whole cell extracts, cells were scraped, lysed with RIPA buffer, and the lysate diluted with an equal volume of buffer containing 2-mercaptoethanol.
Synovial fibroblasts were incubated with rhMIF for 30 minutes, a whole cell lysate was prepared from about 2 × 105 cells by homogenization in the lysis buffer, and the lysate was centrifuged at 14,000 rpm for 15 minutes.
Cells were lysed in NP-40 buffer, and the lysates were incubated with Glutathione Sepharose 4B beads (GE Healthcare) at 4 °C for 2 h, with gentle agitation, and then extensively washed with lysis buffer.
To examine the interaction of optineurin with itself, RGC5 cells or those transfected with pTarget-FLAG-OPTN were lysed in IP buffer and the lysates were subjected to native blue gel electrophoresis.
In brief, after fractionation of cells into the cytoplasmic and nuclear fractions, the nuclear fraction was incubated with RIPA buffer and the lysates were subjected to SDS-PAGE electrophoresis using pre-casted gels from Nupage Bis-Tris (4 12%) gel systems (Invitrogen).
Cells co-expressing HA-hDAT and His-hDATΔEX6, or those co-expressing HA-mGLT1 and His-hDATΔEX6 were homogenized with RIPA buffer, and the lysates were subjected to immunoprecipitation overnight at 4°C with 0.5 µg of anti-His antibody, followed by 1 h of incubation with a 50% slurry of protein-A Sepharose beads (GE Healthcare Biosciences).
After 72 hrs, medium was removed, cells were lysed in 1% Triton X-100 buffer, and the lysates analyzed by on a Fluostar Optima fluorometer (for GFP fluorescence) (BMG Labtechnologies, Durham, NC) and on a 20/20n Luminometer (Turner Biosystems, Sunnyvale, CA) after incubation with firefly luciferase reagent (Promega, Inc., Madison, WI).
