Mouse anti lamin a c

Whole protein extracts were obtained by treating cell pellets with lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5 mM EGTA, 0.25% NP-40, 1 mM DTT, 0.5 mM PMSF, 0.5 mM Na₃VO₄ and protease inhibitor cocktail, Roche). Samples were loaded onto pre-cast 4–12% gradient acrylamide gels (NuPage, Invitrogen). After electro-blotting, filters were incubated for 1 h with mouse monoclonal anti-AKTIP (1 : 400, Sigma), goat anti-actin-HRP conjugated (1 : 3500, Santa Cruz), mouse anti-lamin A/C (1 : 200, Santa Cruz), goat anti-lamin B1 (1 : 200, Santa Cruz), mouse anti-FLAG-HRP (1 : 1000; Sigma), rabbit anti-matrin 3 (1 : 1000, Santa Cruz), goat anti-importin 7 (1 : 200, Santa Cruz), mouse anti-trimethyl H3K9me3 (1 : 350, Millipore) and rabbit anti-TRF2 (1 : 3500, Novus Biologicals). Filters were then incubated with appropriate HRP-conjugated secondary antibodies (Santa Cruz; diluted according to the manufacturer's instructions), which were detected using the enhanced chemiluminescence system (ECL Plus, Amersham). Bands were imaged with the ChemiDoc MP imager (Bio-Rad) and band intensities were quantified using the Image Lab software (Bio-Rad).

Cells were lysed in an extraction buffer consisting of 62.5 mM Tris, 2% SDS, and 2 mg/ml protease and phosphatase inhibitors 18 (Complete, Roche Molecular Diagnostics, pH 7.4). Thirty micrograms of total protein was separated by SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF-FL) membranes (Millipore). Membranes were incubated overnight with the primary antibody at 4°C, rabbit anti-LC3 (1 : 2000, MBL PD014, Nagoya, Japan), rabbit anti-lamin B1 (1 : 1000, Abcam 32454, Cambridge, MA, USA), mouse anti-lamin A/C (1 : 1000, Santa Cruz Biotechnology 376248, Dallas, TX, USA), and mouse anti-β-actin (1 : 10000, Santa Cruz Biotechnology 47778, Dallas, TX, USA) diluted in TTBS/BSA 3%. Following three washes with TTBS secondary antibody IRDye® 680RD goat anti-rabbit (925-68071, LI-COR) or IRDye® 800CW goat anti-mouse (925-32210, LI-COR) that was applied at 1 : 5000 dilution in TTBS, membranes were scanned and analyzed using an Odyssey® IR scanner and Odyssey® Image Studio software 5.2.5.

Cells were washed with ice-cold PBS, lysed in Laemmli buffer (4% SDS, 20% glycerol, and 120 mM Tris-HCl [pH 6.8]) and then incubated for 5 min at 95°C. The DNA was sheared by syringing the lysates 10 times through a 25-gauge needle. Absorbance at 280 nm was measured (NanoDrop; Thermo Fisher Scientific) to determine protein concentration. Samples were prepared using Protein Sample Loading Buffer (LI-COR, #928-40004) and DTT (final concentration 50 mM) and heated at 95°C for 5 min. Proteins were separated using NuPAGE 4-12% Bis-Tris gels (ThermoFisher) and NuPAGE MES SDS running buffer (Thermo Fisher, #NP0002) and transferred to nitrocellulose membranes for immunoblotting. Membranes were blocked in 5% milk PBS and incubated overnight at 4°C with primary antibodies. Next day, membranes were incubated for 1 h at room temperature with IRDye-conjugated secondary antibodies (LI-COR) and scanned on an Odyssey imaging system. The following primary antibodies were used: mouse anti-Tubulin (Sigma Aldrich, #T9026, 1/2000), mouse anti-lamin A/C (Santa Cruz, #sc-7292, 1/500), mouse anti-lamin B1 (Santa Cruz, #sc-365214, 1/1000), rabbit anti-emerin (Proteintech, #10351-1-AP, 1/1000), rabbit anti-H3K9me3 (Abcam, #ab8898, 1/1000), rabbit anti-BAF (ProSci, #4019, 1/500), mouse anti-γH2AX (Millipore, #05-636-I, 1/500) and rabbit anti-H2AX (Bethyl, A300-083A-T, 1/1000).

HEK293 cells growing in 10 cm dish for 48 h were given indicated treatments for 15 min including temperature shifts (37 °C and 25 °C); extracellular pH changes (10 mM HCl and 2 mM NaOH); or changes in external osmotic pressure (250 mOSM/kg or 350 mOSM/kg adjusted through H₂O or NaCl). Cells were then permeabilized using 3 ml of a physiologically isotonic solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose and 10 mM MOPS (pH 6.0 for conditions: 37 °C, extracellular pH 6.0, osmotic pressure 250 mOsm/kg; pH 7.2 for conditions: 25 °C, extracellular pH 7.6, osmotic pressure 350 mOsm/kg)) containing 160 μg/ml digitonin (Sigma) for 3 min. Nuclei were pelleted by centrifugation at 300× g for 5 min and supernatants containing cytoplasmic proteins were collected. Western blotting was performed following electrophoresis on a 10% SDS-polyacrylamide gel. The antibodies used for immunoblotting were rabbit anti-Smad5 (1:1 000, Cell Signaling, 9517), mouse anti-Lamin A/C (1:500, Santa Cruz, SC-7292), mouse anti-β-Tubulin (1:4 000, Sigma, T5201).