Protein phosphatase inhibitor-2

PC3-LN4 cultured cells were transfected as described above for TUNEL analysis. Cells were split as needed and collected at 24, 48, and 72 h post-transfection using trypsin. The cell pellets were frozen at −20°C immediately following collection. For immunoblot analysis, cell pellets were resuspended in 150 μl of RIPA lysis buffer (150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 1% Igepal CA630, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 10% glycerol) with the addition of Sigma protease (P8340) and phosphatase (P2850) inhibitor cocktails (used 1:100). The lysate was frozen at −70°C for 10 min, thawed on ice, vortexed for 5 s, and centrifuged for 5 min at 18,000×g and 4°C in a microcentrifuge. The supernatant was transferred to a fresh tube on ice, and quantitated using Bio-Rad protein assay reagent (500-0006). Equal quantities of lysates were separated in a 10% Tris–Glycine–SDS polyacrylamide gel by electrophoresis, and transferred to nitrocellulose membrane (Whatman 10 402 495) using the wet transfer method in Tris–Glycine–20% methanol. The membranes were blocked for 30 min with 5% nonfat milk (Bio-Rad 170-6404) in Tris-buffered saline (TBS, pH 7.4) with 0.1% Tween 20 (TBS-T) at room temperature. The membranes were then incubated overnight rocking at 4°C in 5% milk/TBS-T containing primary antibody. After washing 3 × 10 min in TBS-T, the membranes were incubated with secondary antibody in 5% milk/TBS-T for 1 h at room temperature and washed in TBS-T as before. Proteins were detected by enhanced chemiluminescence using Pierce SuperSignal West Pico and Dura substrates (Pierce 34078, 34076) and Blue Lite autorad film (ISC BioExpress F-9024-8×10).
Approximately 0.1 mg of tumor tissue was minced then homogenized into 1 ml of cytoskeleton (CSK) buffer and nuclear matrix was prepared as previously described using method C [18 (link)] with the following modifications. The homogenates were not filtered, and the final nuclear matrix pellets were resuspended in 250 μl RIPA lysis buffer and incubated on ice with repeated vortexing for 30 min. The suspension was centrifuged for 10 min at 18,000×g and the supernatant was transferred to a fresh 1.5 ml tube. Quantitations of cytosol and nuclear matrix were performed with the addition of H₂O₂ as described previously [63 (link)]. Cell fractions (20 μg of cytosol, 15 μg of nuclear matrix) were precipitated by addition of 3–5 volumes of −20°C acetone/methanol (1:1), vortex mixing, and centrifugation for 15 min at 18,000×g and 4°C. The pellet was washed with 0.5 ml −20°C methanol and centrifuged for 10 min at 18,000×g and 4°C. The protein pellet was resuspended in 20 μl of Novex loading buffer with tris(2-carboxyethyl)phosphine (TCEP) in RIPA buffer (each diluted to 1× concentration), the samples were heated at 37°C for 10 min, vortexed, and electrophoresed through NuPage 4-12% Bis–Tris 20 well gels (Invitrogen WG1402A) using MOPS SDS running buffer (Invitrogen NP0001). The gels were transferred to nitrocellulose membrane using the wet transfer method in Tris–Glycine–20% methanol. Protein detection was performed as above.
Antibodies used were Actin (sc-1616) and LDH-A (sc-27230) from Santa Cruz Biotechnology; CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; Casein Kinase II α/α′ from BD Transduction Laboratories (611611); CK2β (218712) from Calbiochem; Lamin B₁ (33-2000) from Invitrogen; NF-κB p65 (610868) from BD Transduction Laboratories; Akt (9272) and Phospho-Akt Ser473 (9271) from Cell Signaling. Please note that in Fig. 1a, a chicken polyclonal anti-CK2αα′β antibody was used [64 (link)], however, because following this experiment this antibody was no longer functional, all subsequent immunoblots shown for CK2αα′ used the Bethyl Laboratories antibodies.