Enhanced chemiluminescence detection

After transfection, cells were washed with cold PBS before being solubilized in a lysis buffer containing 120 mM Tris/Hepes, pH 7,4; 150 mM NaCl, 5 mM EDTA, 3 mM KCl; 1% (v/v) Triton X-100 and protease inhibitors (Complete Roche 1697498, Meylan, France). Samples were then harvested and centrifuged at 16,000 rpm for 15 min at 4 °C. For immunoprecipitation, cells were solubilized with lysis buffer containing 0.4 M NaCl; 1.5 mM MgCl₂; 10 mM Hepes, pH 7.9; 5% (v/v) glycerol; 0.5% (v/v) Nonidet P-40) and protease inhibitors (Complete, Roche Diagnostics). Immunoprecipitation was carried out using anti-V5 (Invitrogen) or anti-ManIA (Sigma ) antibody, and affinity purification using protein G-agarose beads (Dynabeads, Invitrogen, Paris, France). After incubation with protein G-agarose beads for 1 h at room temperature, the immunocomplex was washed in PBS (Invitrogen). The protein samples were then boiled in loading buffer, run on gradient 7.5% or 10% or 15% SDS-polyacrylamide gels, probed with primary antibodies of interest and horseradish peroxidase-conjugated secondary antibody. Proteins were visualized by enhanced chemiluminescence detection (Thermo Fisher Scientific, Les Ulis, France) according to the manufacturer′s instructions.

Proteins were extracted from mouse femurs adding RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 0.004% sodium azide) to ground bone. Proteins were run on an 8% SDS-PAGE, transferred to nitrocellulose, blocked with milk, and stained with osteocalcin (15 kDa) primary antibody (Clontech), HRP-linked anti-rat IgG secondary antibody (Abcam) and collagen 1 alpha 2 (130 kDa) primary antibody (Santa Cruz Biotechnology), HRP-linked anti goat IgG secondary antibody (Santa Cruz Biotechnology) and followed by enhanced chemiluminescence detection (Thermo Scientific). The loading control used was β-actin (43 kDa) (Abcam). P-values were calculated using Student’s t-test. Differences with a P-value of <0.05 were considered significant. Data were expressed as mean±SEM (standard error of the mean).

Cell lysates were prepared using RIPA buffer (Thermo Scientific) with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche, Basel, Switzerland), incubated for 15 min on ice and centrifuged at 16 800 g for 10 min at 4 °C. The bicinchoninic acid (BCA) method (Thermo Scientific, Waltham, MA, USA) was used to determine the protein concentration. Proteins were resolved by SDS/PAGE and transferred to polyvinyl difluoride membrane. After blocking with 5% skim milk, membranes were individually probed with anti‐phospho‐AKT (Cell Signaling Technology, Danvers, MA, USA), anti‐phospho‐ERK (Cell Signaling Technology), anticleaved caspase 3 (Cell Signaling Technology), anti‐PARP (Cell Signaling Technology), anti‐p21 (Cell Signaling Technology), anti‐MDM2 (Cell Signaling Technology), anti‐MDM4 (Cell Signaling Technology), anti‐p53 (Cell Signaling Technology), anti‐PUMA (Cell Signaling Technology), and anti‐actin (Sigma‐Aldrich Corporation) antibodies. The membranes were washed and incubated with horseradish peroxidase‐conjugated secondary antibodies, followed by enhanced chemiluminescence detection according to the manufacturer’s instructions (ThermoFisher).

After transfection, cells were washed with cold PBS before being solubilized in a lysis buffer containing 120 mM Tris/Hepes, pH 7,4; 150 mM NaCl, 5 mM EDTA, 3 mM KCl; 1% (v/v) Triton X-100 and protease inhibitors (Complete Roche 1697498, Meylan, France). Samples were then harvested and centrifuged at 16,000 rpm for 15 min at 4 °C. Protein expression levels were assessed after normalizing and loading equal amounts of total protein for 7.5% SDS-PAGE separation and immunoblotting with the antibodies of interest and horseradish peroxidase-conjugated secondary antibody. The primary antibodies used in this study were mouse anti–Myc (Takara, Clontech, Saint-Germain-en-Laye, France) and anti-Sar1A (Invitrogen). Proteins were visualized by enhanced chemiluminescence detection (Thermo Fisher Scientific, Les Ulis, France) according to the manufacturer′s instructions.

Western blotting was carried out by following standard protocols. Protein (30 μg) was separated by denaturing SDS–PAGE and then transferred on to polyvinylidene difluoride membranes. The membranes were blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and then incubated overnight with the primary antibodies at 4 °C. Then, the blots were incubated with the corresponding secondary antibodies conjugated with HRP at RT for 1 h. Visualization was carried out by enhanced chemiluminescence detection (Thermo Fisher Scientific). Uncropped raw version of various blots is included in Supplementary Fig. 6.

Cells were incubated in lysis buffer (50 mM Tris-Cl buffer pH 7.5 containing 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors) for 20 min on ice. Nuclei were pelleted by centrifugation. The protein concentration in post-nuclear supernatants was determined by the bicinchoninic acid method as recommended by the manufacturer (Sigma), using bovine serum albumin as standard. Proteins from cell lysates or virus purification fractions were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Hybond-ECL; Amersham) by using a Trans-Blot apparatus (Bio-Rad). The proteins of interest were revealed with specific primary antibodies, followed by species-specific secondary antibodies conjugated to horseradish peroxidase (Jackson Immunoresearch), and enhanced chemiluminescence detection as recommended by the manufacturer (Thermofischer).