Anti cd97

At 75–80% confluence, cells were treated with 150 μl of 10x lysis buffer (Cell Signaling Technology) combined with protease inhibitor tablets (Roche), phosphatase inhibitor tablets (Roche), and 1 mM phenylmethanesulfonyl fluoride (PMSF). Samples were run on a 4–20% Tris-Glycine gel (Invitrogen) and transferred to an Immun-Blot PVDF membrane (BioRad). After blocking in 5% nonfat dry milk (BioRad), samples were incubated in the following primary antibodies: anti-CD97 1:500 (Abcam ab108368), anti-nestin 1:4,000 (abcam ab22035), anti-Sox2 1:1,000 (Abcam ab75485), anti-GAPDH 1:10,000 (Cell Signaling 2118S). Secondary antibodies conjugated to horseradish peroxidase were used with the Amersham ECL system (GE Healthcare) to detect protein.

Ammonium bicarbonate (NH₄HCO₃), phenylmethylsulfonyl fluoride (PMSF), formic acid (HCOOH), and iodoacetamide (IAA) were obtained from Sigma (St. Louis, MO). Tris[2-carboxyethyl] phosphine (TCEP) Bond-Breaker^™ was from Pierce (Rockford, IL). Acid-cleavable surfactant: 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS) was from Protein Discovery Inc. (Knoxville, TN). All chemicals used were A.C.S. grade or higher, and all solvents used were HPLC-grade or higher. Sequence-grade modified trypsin was obtained from Promega (Madison, WI). The anti-MCAM (CD146), anti-BCAM (CD239), anti-CD99, and anti-CD97 antibodies used for western blot (WB) were from Abcam (Cambridge, United Kingdom).