Sds sample buffer

Immunoblotting protein samples were prepared for SDS-PAGE in SDS sample buffer (Life Technologies) and boiled at 95 °C for 10 min before electrophoresis on 12% gels. Proteins were transferred to PVDF membranes (Amersham). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies. The following primary antibodies were used at the indicated dilutions: rabbit phospho-c-Jun (Thr91) (Cell signaling, 2303, 1:1000), rabbit anti-c-Jun (Cell signaling, 9165, 1:1000) and rabbit anti-GAPDH (Sigma, G9545, 1:1000). Immunoreactivity was visualised using appropriate HRP-conjugated secondary antibodies. Immunoblots were developed using the ECL Advance Western blotting detection kit (Life Technologies) and exposed to Kodak X-Omat AR films. Image analysis and quantification of band intensities was done with ImageQuant (GE Healthcare).

Multilamellar vesicles (MLVs) were formed from POPC and Chol (1:1 mol:mol). Lipids were dissolved in chloroform and the solvent was evaporated using rotary evaporator. 10 mM MES buffer (pH 5.7) with 150 mM NaCl and 1 mM EDTA was added to dried lipid film which was freeze-thawed three times using liquid nitrogen to form 22 mM MLVs. Proteins (2 µg) were added to MLVs to give a final 1:2500 molar ratio (LLO:lipids). Samples were incubated 30 min at room temperature and then centrifuged for 15 min at 16 100 g. Clear supernatants were collected and pellets were washed once in MES buffer. All samples were then mixed with SDS Sample Buffer (Life Technologies) and denatured for 10 min at 70 °C. Total sample volumes were analysed for protein presence on 4–12% Bis-Tris gels (Life Technologies). As a control, each of the proteins was incubated in MES buffer without MLVs and treated the same. 2 µg of each protein was applied to gels as a control. The samples, derived from the same experiment were applied on 4 separate gels that were processed in parallel. Novex Sharp Unstained Protein Standard was applied twice on each gel and bands (all at 56.4 kDa) were cropped from original gels for final presentations. The gel images were not further processed.

Cells (2 x 10⁶) were washed with phosphate-buffered saline (PBS) and lysed in 100μl of SDS sample buffer (LifeTechnologies). Whole-cell extracts (15μl [from 3 x 10⁵ cells]) were separated on 10% acrylamide gels (LifeTechnologies) or Phos-Tag gel (Wako Chemicals). Following electrophoresis, the gel was transferred to a PVDF membrane using an iBlot Gel Transfer system (LifeTechnologies)and stained using anti-SAMHD1 (Proteintech), anti-tubulin (Sigma-Aldrich), and anti-GAPDH (Santa Cruz Biotechnology) polyclonal antibodies.

In vitro PARylation assays using recombinant His-PARP1 or immunoprecipitated endogenous PARP1 from MEFs were performed as previously described (Zaniolo et al., 2007 (link)). Briefly, PARP1 protein or immunoprecipitant was incubated for 20 min or 2 min at 30°C with GST or GST-Sam68 in a standard assay mixture containing 100 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 10% (v/v) glycerol, 1.5 mM DTT, 10 μg/ml activated DNA (sonicated) and 200 μM NAD⁺. The reaction was terminated by the addition of SDS sample buffer (Life Technologies), and the boiled samples were subjected to SDS-PAGE. When indicated, the PARP inhibitor PJ-34 was added to the reaction mixture at a final concentration of 1 μM for 15 min prior to the reaction.

Cultured matrices were directly dissolved in sodium dodecyl sulfate (SDS) sample buffer (Life Technologies), heated at 80°C for 10 min, and centrifuged at 8,000 × g for 20 min. Equal volumes of supernatant were loaded onto the NuPAGE 3–8% Tris-Acetate gel (Life Technologies), and electrophoresis was performed. Gels were directly stained with Coomassie Brilliant Blue R-250 (CBB) (Wako Pure Chemicals Ind., Ltd., Osaka, Japan) in CBB buffer (10% acetic acid, 50% methanol, and 40% deionized distilled water (DDW)) for 30 min, and de-stained with CBB buffer overnight.