Tissue lysates, immunoprecipitation and Western blotting

Frozen tissues were each pulverized under liquid nitrogen using a pre-chilled mortar and pestle, and powdered tissue was stored at −80C or in liquid nitrogen until use. To prepare lysates, we added powdered tissue (80–100 mg) to 500 uL ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 0.3% v/v Triton-X100, 5 mM EDTA, 1 mM DTT, 100 uM PMSF, 1 ug/mL Pepstatin A, 1X Thermo Scientific Halt Protease Inhibitor Cocktail #78430, 50 nM Thiamet G [OGA inhibitor; 1,2-dideoxy-2′-ethylamino-α-d-glucopyranoso-[2,1-d]-Δ2′-thiazoline; provided by G. W. Hart) and 50 mM UDP-GlcNAc [Sigma]) in a 2 mL Eppendorf tube on ice, and moved to liquid nitrogen as needed to keep frozen until further processing. Thiamet G and UDP-GlcNAc were included to maintain labile O-GlcNAc modifications. Samples were flick-vortexed to mix, incubated 10 min on ice, vortexed 10 s at high speed, then sonicated on ice 20 times (0.5 s bursts) and finally centrifuged 30 min (16,000 g, 4°C) to pellet insoluble material. Sonication greatly facilitates solubilization of lamins and associated proteins (Berk et al., 2013; Berk and Wilson, 2016). Supernatant protein concentrations were measured via Bradford assay and adjusted with lysis buffer to 1 ug/uL before use.

For each preparatory immunoprecipitation, 500 uL lysate (500 ug total protein) was incubated with 10 uL anti-lamin A/C mouse mAb 4C11 (Cell Signaling Technologies #4777; 1:50 dilution) with rotation overnight at 4°C. We then added 10 uL Protein G Sepharose slurry (GE Healthcare #17-0618-01, prewashed three times in 300 uL lysis buffer) to each reaction and rotated 1 h at 4°C. After pelleting (1 min, 13,300 g, 4°C), the beads were washed three times in 300 uL lysis buffer. For mass spectrometry analysis, bound proteins from each sample were eluted using 50 uL 1% SDS. Alternatively, for SDS-PAGE and Western blotting of smaller scale immunoprecipitations, bound proteins were eluted by heating (95°C) for 5 min in 30 uL of 2x SDS-sample buffer.

Immunoprecipitates (20 uL each; corresponding to 40 ug input lysate protein) were resolved on Bolt 8% Bis-Tris Plus gels in MOPS running buffer for 5 min at 200 V (20°C–22°C), then at 170 V for 1.5 h (4°C). Resolved proteins were transferred to nitrocellulose membranes for 1.5 h at 300 mA, at 4°C. Membranes were blocked 1 h in blocking buffer (3% BSA, 0.01% Tween-20, 20 mM Tris Base, 137 mM NaCl, pH 7.6). The primary antibody, which specifically recognizes the O-GlcNAc modification (IgM mAb CTD110.6, Santa Cruz Biotechnologies, 1:1000; provided by Natasha Zachara) was first diluted into blocking buffer. Blots were then rocked overnight (4°C), washed three times with TBST buffer (0.01% Tween-20, 20 mM Tris Base, 137 mM NaCl, pH 7.6), incubated (1 h at 20°C–22°C) with secondary antibody (HRP-conjugated anti-mouse IgM; Santa Cruz Biotechnology #sc-2064; dilution 1:10,000) in blocking buffer, washed three times in TBST, and finally visualized by enhanced chemiluminescence (Hyblot CL autoradiography film #E3012).

To detect bound lamin proteins, we next incubated with lamin A/C antibodies. Blots were not allowed to dry; each blot was stripped for 10 min in stripping buffer (1.5% w/v glycine, 0.1% w/v SDS, 1% v/v Tween-20, pH 2.2), washed twice (10 min each) in PBS (16 mM Na₂HPO₄, 3 mM KH₂PO₄, 270 mM NaCl, 5.4 mM KCl, pH 7.4) and twice (5 min each) in TBST. Blots were then blocked 1 h at 20°C–22°C using 3% BSA in TBST, incubated with lamin A/C antibodies (sc-20861 rAb, Santa Crus Biotechnologies, 1:1000 in blocking buffer) overnight at 4°C, then incubated with secondary antibody (HRP-conjugated anti-rabbit IgG; Cell Signaling Technologies #7074S; dilution 1:10,000), washed, and visualized by enhanced chemiluminescence (Hyblot CL autoradiography film #E3012). Films were scanned with the Epson Perfection V500 Photo scanner. Western blot signals were quantified via Quantity One, version 4.6.9.