Pou2f1

Nuclear and cytoplasmic protein was extracted from cells by suspension in a solution of Buffer A containing 10 mM HEPES pH 7.4, 1.5 mM MgCl₂, 10 mM NaCl, 0.1% NP-40 and a cocktail of protease inhibitors (Complete Mini Cocktail, Roche Diagnostics Ltd, Lewes, UK). The cells were lysed on ice for 10 min after which they were passed through a 21 gauge needle to ensure complete plasma membrane lysis. Nuclei were pelleted by centrifugation (12,000 x g at 4°C for 2 min) and the supernatant containing cytoplasmic protein was retained. Nuclei were washed in a solution of Buffer A (as specified above). The nuclei were lysed for 10 min on ice in Buffer A and sonicated for 30 sec to completely disrupt the nuclear membrane. Remaining cell debris was pelleted by centrifugation and the supernatant containing nuclear protein retained. Total protein content was determined by the Bradford protein method using the BCA protein assay kit (Pierce, Cramlington, UK). Protein (30 μg) was loaded onto a Tris-Glycine polyacrylamide gel (10%) and subsequently transferred to a nitrocellulose membrane. The antibodies used for Western blotting were β-tubulin (Abcam 1:1000), HOXD10 (Biorbyt 1:200), POU2F1 (Abcam 1:1000) and TATA-BP (Abcam 1:1000).

The specific reagents included the primary antibodies against AKT, POU2F1, and TRIM21 (Abcam, London, UK); against PI3K^p100α, γ-H₂AX, 53BP1 and p-AKT^Ser473 (Cell Signaling Technology, Danvers, MA, USA); against α-tubulin, BCL-2 and BAX (Proteintech, Chicago, USA); horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Beyotime, Shanghai, China). GSK2795039, 3-MA and DADS (oil, ≥98%, and 1.008 g/mL) were purchased from Sigma, (Saint Louis, Missouri, USA), and the DADS was fully dissolved in Tween 80 and diluted at 1:100 in physiological saline and stored in a -20°C freezer. Additionally, 740Y-P, LY49002, MG132, AG activator 1 and cycloheximide as well as AV-153, an antimutagenic molecule were obtained from Selleckchem, Houston, USA and MedChenExpress, Monmouth Junctio, USA, respectively.