Laemmli buffer

Protein lysates from liver and epididymal white adipose tissue (WAT) were homogenized in RIPA buffer (Sigma–Aldrich) supplemented with 1% protease inhibitor Cocktail (Thermo Fisher Scientific). Lysates were centrifuged for 10 min, 10,000 g, at 4 °C and supernatants were used for Western blot analysis. Total protein concentration was determined by Bicinchoninic Acid Kit for Protein Determination (Thermo Fisher Scientific). 20 μg of total cellular protein was diluted 1:1 with 2x Laemmli buffer (Bio-Rad) and denatured at 95 °C for 5 min. MSC protein lysates were directly homogenised in 2x Laemmli buffer (Bio-Rad). Final protein lysates were resolved by SDS-PAGE 7% and transferred to PVDF membranes in 20% methanol, 200 mM Gly, 25 mM Tris, pH 8.3. The membrane was blocked for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies. The blots were washed and exposed to horseradish peroxidase-labeled secondary antibody (1:10,000) for 1 h at room temperature. The blots were then washed and the immunocomplexes visualized by the chemiluminescence detection system SuperSignal West Pico PLUS Substrate (Thermo Fisher Scientific).

OMVs were collected as described previously^{8 (link)}. Briefly, overnight cultures were diluted 1:100 into fresh LB and grown to OD₆₀₀ = 0.6-0.8. To collect whole-cell lysate controls, an OD₆₀₀ = 1 culture was obtained by centrifugation, resuspended in 50 µL of 2X Laemmli buffer (BioRad, Cat. #1610747) with 10% β-mercaptoethanol (MilliporeSigma, Cat. #M3148), and boiled for 10 min. To collect OMVs, an OD₆₀₀ = 3 culture was obtained by centrifugation. Supernatants were collected and adjusted to equal volume with fresh LB. Supernatants were filtered through a 0.2-μm filter (MilliporeSigma, Cat. #SLGPR33RB) and then through an Amicon Ultra-15 centrifugal filter with 100 kDa molecular weight cutoff (MilliporeSigma, Cat. #UFC9100) to isolate and concentrate OMVs. Centrifuged samples were adjusted to equal volume and resuspended in 2X Laemmli buffer (BioRad, Cat. #1610747) supplemented with 10% β-mercaptoethanol (MilliporeSigma, Cat. #M3148). Samples were boiled for 10 min, loaded on a 10% SDS-polyacrylamide gel, and processed as described above by immunoblot analysis.

Chromatin fractionation was performed as described [38 (link), 61 (link)]. The preparation was carried from duplicate samples to enable presentation of all fractions. Approximately 1 × 10⁷ cells were washed in PBS and resuspended in 200 μl of solution A (10 mM Hepes [pH 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, and protease and phosphatase inhibitors). Triton X-100 was added to a final concentration of 0.1%, cells were incubated on ice for 5 min, and the cytoplasmic (S1) and nuclear fractions (P1) were harvested by centrifugation at 1,300 × g for 4 min. Isolated nuclei were then washed in solution A, lysed in 150 μl solution B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and protease and phosphatase inhibitors), and incubated on ice for 10 min. The soluble nuclear (S2) and chromatin fractions (p2) were harvested by centrifugation at 1,700 × g for 4 min. Isolated chromatin was washed once with solution B and spun down at high speed (10,000 × g for 1 min). Finally, chromatin was resuspended in 150 μl of SDS sample buffer and sheared by sonication. Protein concentrations were determined by BCA assay (ThermoScientific, Rockford, IL). Equal amounts of protein from whole cell extracts or from the different cellular fractions were mixed with 5 × laemmli buffer (Bio-Rad) and analyzed by immunobloting.

Cells were lysed in RIPA buffer [50 mM tris (pH 7.4), 1% triton, 0.5% NA‐DCA, 0.1% SDS, 150 mM NaCl, and 2 mM EDTA] with Halt™ protease (Thermo Fisher Scientific, A32955) and phosphatase (Thermo Fisher Scientific, 78,428) inhibitor cocktails (N = 3). Proteins were isolated, and the concentration was assessed by DC protein assay (Bio‐Rad, 500–0116). Lysates were eluted in 5× Laemmli buffer (Biorad, 1,610,747) supplemented with 0,1 M DL‐Dithiothreitol (Sigma, D0632‐1 g). Equal amounts of proteins were separated by SDS‐PAGE and transferred onto polyvinylidene difluoride membranes (Biorad, 1,704,156). Filters were washed in TBS + 0.05% Tween (Brunschwig, SER42598‐01) and blocked for 1 h with 5% non‐fat milk (Applichem, A0830‐1 kg). Primary antibodies used are the following: Anti‐phospho‐ubiquitin (Ser65) (Millipore, ABS1513‐I, 1:1000); Tom20 (Santa Cruz, sc‐17,764, 1:1000); and HSP90 (Proteintech, 60,318‐1‐Ig, 1:3000).