Anti lamin a c

Immunoblotting and immunoprecipitation were performed as previously described^{52 (link)}. Anti-MSK1, anti-Aurora B, anti-β-actin, anti-NFATc2, anti-GADPH, anti-α-tubulin, anti-laminA/C were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-H3S10 and anti-H3 were from Active Motif (Carlsbad, CA, USA). Anti-STAT3, anti-AKT, anti-p42/44, anti-p-STAT3 Y705, anti-p-STAT3 S727, anti-p-AKT S483, anti-p-p42/44 were from Cell Signaling Technology (Danvers, MA, USA).

Proximity Ligation Assay (PLA) experiments was performed using kits from Sigma-Aldrich: Duolink® in situ Detection Reagents Orange (DUO92007). Briefly, methanol-fixed cells were saturated with saturated 4%-BSA and incubated with anti-lamin A/C (Santa Cruz sc-376248) and anti-P53BP1 (Cell Signaling 4937) primary antibodies overnight at 4°C. Thereafter, slides were incubated for 1 h at 37°C with secondary antibody-conjugated PLA probe. Ligation solution was added to each sample and slides were incubated in a humidity chamber for 30 min at 37°C. Ligation solution was removed with washing buffer and amplification solution was added. Slides were incubated in a humidity chamber for 100 min at 37°C and then washed with wash buffers. DNA was counterstained with DAPI and samples were observed by a Nikon Eclipse Ni fluorescence microscope equipped with a digital CCD camera and NIS Elements AR 4.3 software. Quantitative analysis was performed using Duolink Image Tool software (Sigma) by counting 300 nuclei per sample.

Immunoprecipitation and imunoblotting were performed as described previously [26 (link)]. The reagents were as following: anti-β-catenin (BD Bioscience, Cat# 610153), anti-γ-catenin (BD Bioscience, Cat# 610253), anti-CBP (Santa Cruz, clone SC-369), anti-p300 (Santa Cruz, clone SC-584), anti-activated- β-catenin (Millipore, clone 8E7), anti-lamin A/C (Santa Cruz, SC-7293), NE-PER Nuclear extraction reagent (Pierce, Cat#78833), Protease inhibitor cocktail (Calbiochem, Cat#539137), Protein A-agarose (Roche, Cat#11134515001), Illustra microspin columns (GE Healthcare, Cat#27-3565-01), ECL Plus (GE Healthcare, Cat#RPN 2132), and Blue ultra autorad film (BioExpress, Cat# F9029-8X10).

Primary antibodies used in this study were anti-beta actin (Abcam, ab8224), anti-ERK1/2 (Cell Signaling, 9102), anti-phospho ERK1/2 (Cell Signaling, 9101), anti-lamin A/C (Santa Cruz, sc-7292), anti-lamin B1 (Santa Cruz, sc-30264), anti-S6 kinase (Cell Signaling, 2317), anti-phospho S6 kinase (Cell Signaling, 2211) and anti-LAP1 (Atlas antibodies, HPA050546).

Fibroblasts were seeded into 4 chamber-well slides (SPL Lifesciences, Korea). Cells were fixed for 15 minutes (RT) in a 4% paraformaldehyde +2% sucrose solution and then permeabilized for 3 minutes at RT using permeabilization buffer (0.5% Triton X-100, 50 mM NaCl, 300 mM sucrose, 20 mM HEPES pH 7.5, 3 mM MgCl2). Cells were then incubated with primary antibodies for 40 minutes at 37 °C (anti-progerin (1/50, sc-81611) and anti-lamin A/C (1/50, sc-20681), Santa Cruz Biotechnology). After washing, cells were incubated for 20 minutes at 37 °C with secondary antibodies (A11001, A11012, 1/400 Life Technologies). Nuclei were stained with DAPI for 10 minutes at room temperature (0.1 μg/mL, Thermofisher). Slides were mounted using FluorSave™ reagent (Merck Millipore) and observed on a fluorescence microscope (ApoTome.2 Zeiss).

The 4-hydroxynonenal was obtained from Biomol (Plymouth Meeting, PA, USA). Dulbecco's modified essential medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO Invitrogen (Carlsbad, CA, USA). The following antibodies, anti-COX-2, anti-Lamin A/C, and anti-beta actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Pim-2 and myc-tagged antibodies were from Millipore (Billerica, MA, USA). The pp70S6K, p70S6K, p4EBP1, 4EBP1, pBad, Bad, NF-κB (p65), and GAPDH antibodies were from Cell Signaling Technology (Danvers, MA, USA). Other chemicals were of the highest purity available.