Mouse anti lamin b1

Cells or mouse tissues were lysed with RIPA buffer (20-188; Sigma-Aldrich) supplemented with combinations of protease/phosphatase inhibitors (APExBIO). Around 40–60 μg protein was separated with gradient SDS–PAGE gel (4–20%, ACE Biotechnology). The following primary antibodies were used: rabbit anti-UPF3A+UPF3B (ab269998, Abcam, 1:1,000); mouse anti-β-actin (A5441; Sigma-Aldrich, 1:10,000); mouse anti-Lamin B1 (sc-374015; Santa Cruz). The secondary antibodies used in these studies were HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:2,000; Proteintech).

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).

The mouse antibody anti-H3K9me2/3, and the rabbit antibodies anti-H3K9ac, -H3K4me3, -H4K20me3, -H3 and -G9a were from Cell Signaling (Beverly, MA, USA). Rabbit anti-GLP was from Thermo- Scientific (Waltham, MA, USA). Mouse anti-lamin B1 (for the HMT experiment) was from Santa Cruz Biotechnology (Dallas, TX, USA) and rabbit anti-lamin B1 was from Abcam (Cambridge, UK). The rabbit antibody against suv39h1 was from Abcam and the mouse anti-β-tubulin was from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD3 and anti-CD28 were from Biolegends (San Diego, CA, USA). 12G10 (anti-β1 activator Ab), 9EG7 (anti-β1 activator Ab) and mab13 (anti-β1 blocking Ab) were kindly provided by Martin Humphries (University of Manchester, UK). VCAM1 was obtained from Martin Humphries and Peprotech. Fibronectin fragment FN-H50 and FN-H120 were kindly provided by Martin Humphries. Fluorescence-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). ICAM1, IL-4, CXCL12 and CCL19 were from Peprotech (Rocky Hill, NJ, USA). BIX01294, chaetocin, actinomycin were from Sigma-Aldrich. Hoechst 33342 was from Invitrogen (Grand Island, NY, USA). CFSE and Cell Tracer Far Red were from Life Technologies (Paisley, UK).