Anti skp2 2c8d9

Two serial sections of two tissue microarrays containing human prostate tumor tissues were subjected to Ventana autostainer model Discover XT ™ (Ventana Medical System, Tuscan, AZ). The primary antibodies were anti-DAB2IP [23 (link), 24 (link)]and anti-Skp2 (2C8D9) from Zymed, San Francisco, CA. Two pathologists assessed and scored the immunostaining independently and reached a final consensus for any inconsistent scoring. Briefly, values on a four-point scale were assigned to each specimen. The intensity score was assigned, which represented the average intensity of positive cells (0, none; 1, weak or questionably present stain; 2, intermediate intensity in a minority of cells; and 3, strong intensity in a majority of cells). High expression was defined as score higher than average, and low expression was defined as score lower than average.

PNT1A, PC3, LNCaP, PZ-HPV-7, C4-2, LAPC4, 293 and their sublines were maintained as described previously [22 (link)-24 (link), 32 (link)]. Anti-DAB2IP polyclonal antibody was used for western blot analysis and IHC as described previously [24 (link), 32 (link)]. Anti-FLAG-HRP (M2) was obtained from Sigma (St. Louis, MO). Anti-Skp2 (sc74477), anti-Ubiquitin (sc271289), anti-Tubulin (32239), anti-Akt 123 (H36, sc8312), and anti-GAPDH (sc16674) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Akt (Ser473) polyclonal antibody (#9271) was from Cell signaling Technology (Danvers, MA). Anti-p21 (6B6) and Anti-p27 were obtained from BD Pharmingen (Sparks, MD). Anti-Skp2 (2C8D9) was from Zymed (South San Francisco, CA) Proteasome inhibitor MG132 was purchased from Calbiochem (Gibbstown, NJ), cycloheximide and 2-(4-morpholinyl)-8-phenyl-chromone (LY294002) were also purchased from Sigma. For cDNA transfection, cells were seeded in plates with 70-80% confluence before transfection. The transfection was carried out using Lipofectamine LTX with Plus™ reagent (Invitrogen, Carlsbad, CA) or polyethylenimine (PEI, Polysciences Inc., Warrington, PA) according to the manufacturer's instructions.