Emerin

Protein extraction from cells was accomplished using RIPA buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 10% sodium deoxycholate). Samples were separated on SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane. Blotted membranes were blocked with 3% skim milk for 1 h followed by incubation with specific primary antibodies. The following antibodies were used in this study: Lamin A/C (sc-376248), GST (sc-138), green fluorescent protein (GFP) (sc-9996), p53 DO1 (sc-126), Actin (sc-47778), His (sc-8036), nuclear factor-κB (sc-372), IκBα (sc-371), and p21 (sc-397) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); α-FLAG (F3165) from Sigma (St. Louis, MO, USA); Emerin from Novocastra (New castle, UK); PUMA (4976), phospho-Erk (9101), total-Erk (9102), phospho(S15)-p53 (9286), phospho(S20)-p53 (9287), and BMI-1(5856) from Cell Signaling Technology (Danvers, MA, USA); NOXA (ALX 804-408-C100) from Enzo Life Sciences (Farmingdale, NY, USA); p16 (10883-1-AP) from Proteintech (Rosemont, IL, USA); and Histone H3k9me3 (ab8898) from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit, and mouse anti-goat IgG antibodies (Pierce, Thermo Fisher Scientific, Inc., Rockford, IL, USA) were used as secondary antibodies.

Primary antibodies used were as follows: prelamin A (SC-6214, C-20), lamin A/C (SC-6215, N-18), FLAG (M2, F3165) (Sigma); γ-H2AX (2577), PCNA (PC10) (Cell Signaling Technology, Danvers, Mass); Emerin (Novocastra); Kap1 (ab70369), γ-Tubulin (ab11316), Pol η (ab17725) and (ab180703), USP3 (ab82935), Thymine dimer (ab10347), BrdU (ab6326), H2AX (ab11175) (abcam); Ki-67 (VP-K452) (Vector Laboratories).
For immunofluorescence, cells were cultured on coverslips and were fixed in 4% paraformaldehyde in PBS followed by 3 min permeabilisation with 0.5% NP-40 in PBS or 100% methanol at −20°C for 10 min. For BrdU analysis, cells were incubated in 2M HCl for 40 min at room temperature (RT) prior to permeabilisation. Coverslips were then incubated with blocking solution (3% BSA in PBS) for 1 h at RT. Primary antibodies in blocking solution were then applied for 12 h at 4°C, followed by 1 h RT incubation with fluorescent dye conjugated secondary antibodies (Invitrogen). Coverslips were washed with PBS, mounted onto slides with medium containing DAPI, and visualized using a Leica SP5 confocal microscope.