A0545

Frozen specimens of mesenteric fat were homogenized in 20 mM of Tris–HCl buffer (pH 7.8) containing 0.2 % of Triton X-100 and protease inhibitor cocktail (Sigma, USA), and centrifuged. Aliquots of supernatant (10 μg of protein) were separated by 10 % of SDS-PAGE and electroblotted onto Immuno-Blot™ PVDF Membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked by incubation with a blocking buffer and then incubated with rabbit polyclonal anti-HSL antibody (SCB45041763, Sigma-Aldrich USA), polyclonal anti-phospho-HSL antibody (pSer⁵²² in human/Ser⁵⁶³ in rat; SAB4501763, Sigma-Aldrich USA), anti-ABHD5 antibody (AV42055, Sigma-Aldrich USA), anti-ATGL antibody (sc-67355, Santa Cruz Biotechnology USA), anti-G0S2 antibody (sc-133424, Santa Cruz Biotechnology USA), and anti-actin antibody (A0545, Sigma-Aldrich USA). The secondary HRP-conjugated antibodies were obtained from Sigma Aldrich (A0545). The reactions were visualized with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Inc., Rockford, IL, USA). The bands visualized on the film following chemiluminescent detection were compared with the molecular mass protein markers visible on the membrane after electroblotting (SM26634, Fermentas). The film was adjusted to the membrane in such way that the membrane edges were visible on the film.

To detect the phosphorylation status of S6K, 50 mg plant aerial tissue was used for protein extraction using 1.5 mL extraction buffer of 1 × PBS, pH 7.4, containing 250 mM sucrose, Protease Inhibitor Cocktail (Sigma-Aldrich, P9599) and PhosSTOP phosphatase inhibitor (Roche, 4906845001). Three times of centrifugation, 1 k × g for 5 min, 14 k × g for 5 min and 135 k × g for 30 min, were conducted to separate the soluble proteins. The supernatant from the last centrifugation was separated, concentrated to 200 μL using an Amicon Ultra centrifugal unit (Millipore, UFC501024), and then mixed with 40 μL 6 × Laemmli buffer. Proteins were denatured by incubation at 95°C for 10 min. Protein samples were separated on 15% SDS-PAGE with 8M urea and blotted to PVDF membranes (Bio-Rad, 1620177). Blots were blocked with 5% milk for 1 hr at room temperature. Blots were incubated with primary antibodies of either anti-S6K (Agrisera, AS12 1855) or anti-S6K-phosphorylated (Abcam, ab207399) overnight at 4°C and subsequently with secondary HRP conjugated goat anti-rabbit antibody (Sigma-Aldrich, A0545) for 1 hr at room temperature.

All experimental procedures in this section were performed according to previously described methods [48 (link), 49 (link)]. Briefly, gene expression in gmNZ9000 was induced with 1.25 ng/mL nisin (MoBiTec) for 3–4 h in 2-mL cultures. Bacterial cells and supernatants were separated by centrifugation, and protein samples were prepared. These samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10 % (v/v) polyacrylamide). Electrophoresed proteins were visualized by gel staining with Coomassie Brilliant Blue or transferred from the gel onto a polyvinylidene difluoride membrane for western blotting. Western blotting was performed with mouse anti-His-tag Ab (1/1000) (652501; BioLegend, San Diego, CA, USA) or rabbit anti-HO-1 Ab (1/1000) (SAB2101053; Sigma-Aldrich, St. Louis, MO, USA), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Ab (1/5000) (A4416; Sigma-Aldrich) or HRP-conjugated goat anti-rabbit IgG Ab (1/5000) (A0545; Sigma-Aldrich). The experiments were repeated three times.