Purified mouse anti human retinoblastoma protein clone g3 245

Portions of harvested tumors were resuspended at 100 mg/mL in RIPA buffer supplemented with protease and phosphatase inhibitors. Next, we homogenized these tumor samples with pestles in tubes, and then sonicated the samples for 10 s at 50% amplitude on a Fisherbrand Model 120 Sonic Dismembrator. Cells cultured on dishes were collected by scraping in 1× PBS and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Proteins from lysates were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane using the iBlot 2 dry blotting system (Invitrogen IB21001).
Membranes were incubated overnight at 4°C with the following antibodies: Phospho-Rb (Ser807/811) (D20B12) XP Rabbit mAb (Cell Signaling Technology #8516), Purified Mouse Anti-Human Retinoblastoma Protein Clone G3–245 (BD Biosciences 554136), Monoclonal ANTI-FLAG M2 antibody produced in mouse (Sigma-Aldrich F1804), E2F-1 Antibody (Cell Signaling Technology #3742), and mouse monoclonal β-Actin Antibody N-21 (Santa Cruz Biotechnology sc-130656). The primary antibodies were detected using the fluorescently labeled secondary antibodies IRDye 680LT Goat anti-Mouse IgG (LI-COR 926–68020) and IRDye 800CW Goat anti-Rabbit IgG (LI-COR 926–32211). Membranes were imaged on a LI-COR Odyssey CLx and analyzed with LI-COR ImageStudio software.