Branson sonifier 450

Tissue sample homogenization and sonication were carried out by means of the Wheaton® 903475 Overhead Stirrer apparatus (Wheaton, Millville, New Jersey, USA) and Branson Sonifier 450 (Branson Ultrasonics, Danbury, USA), respectively. Total protein concentration was determined in duplicate by Bradford assay (Bio-Rad Laboratories, Hercules, California, USA) by means of a UV-Vis spectrophotometer (8453 UV-Vis Supplies, Agilent Technologies, Waldbronn, Germany) using BSA as the protein of reference. HPLC-ESI-MS/MS analyses were performed on an UltiMate 3000 RSLCnano System coupled to an Orbitrap Elite MS detector with EASY-Spray nanoESI source (Thermo Fisher Scientific). EASY-Spray columns 15 cm × 50 μm ID, PepMap C18 (2 μm particles, 100 Å pore size), and 15 cm × 75 μm ID, PepMap C18 (5 μm particles, 300 Å pore size) (Thermo Fisher Scientific), were used for bottom-up and top-down analyses, respectively, in coupling to an Acclaim PepMap 100 cartridge (C18, 5 μm, 100 Å, 300 μm i.d. × 5 mm) (Thermo Fisher Scientific).

The cells grown under each culture condition were washed by cold phosphate‐buffered saline and were directly harvested with 1× SDS sample buffer. The cells were sonicated by Branson Sonifier 450 (Branson Ultrasonics, Danbury, CT, USA). The homogenate (5–20 μL of each sample) was separated on SDS/PAGE gels for western blotting with anti‐DGKδ [9 (link)], anti‐cyclin D1 (sc‐450, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐cyclin D3 (sc‐182, Santa Cruz Biotechnology), anti‐myogenin (sc‐12732, Santa Cruz Biotechnology), anti‐myosin heavy chain (MyHC; MF20, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti‐β‐tubulin (T4026, Sigma–Aldrich), anti‐β‐actin (A5441, Sigma–Aldrich) and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; 016‐25523, Wako Pure Chemicals) antibodies. The immunoreactive bands were visualized using horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse IgG antibody (Cell Signaling Technology, Danvers, MA, USA) and Pierce™ ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA). The intensity of each band was measured using Amersham™ ImageQuant™ 800 (GE Healthcare, Chicago, IL, USA).

Thoroughly washed biomass pellet were suspended in 100 ml of sterile water and sonicated in a Branson Sonifier 450 (Branson Ultrasonics Corp., USA) for 3–5 min at maximum output and duty cycles to ensure complete breakage of the cells. If the extract showed the presence of filaments/cells, steps of sonication was repeated. The resulting extracts were centrifuged at 10,000 g for 30 min, filtered through Whatman No. 1 filter paper and stored in refrigerator at 4 °C for further use as cell extract^{18 (link)}. The chemical profile of cyanobacterial extract was analysed using GC–MS to find out the probable compounds that have reducing potential and aided the synthesis of nanoparticles. Samples for GC–MS analysis were prepared by dissolving the dried extract in methanol. The GC–MS analysis was done by Shimadzu GC–MS QP 2010 Plus equipment in electron ionization (EI) mode fitted with a RTX-5 capillary column (60 m × 0.25 mm × 0.25 μm). Helium was used as carrier gas with 0.7 ml min^–1of flow rate. The temperature of the injector was fixed at 260 °C. The initial and final temperature of the column was 80 °C and to 280 °C at the rate of 10 °C min^–1 and 15 °C min^–1 respectively. A 3.5 min solvent delay was used. Mass spectra were recorded under scan mode in the range of 40–650 m/z. Compounds were identified by comparing with NIST11/ WILEY library^{81 (link)}.

Tissue homogenization and sonication were carried out by means of a Wheaton^® 903475 Overhead Stirrer apparatus (Wheaton, Millville, NJ, USA) and a Branson Sonifier 450 (Branson Ultrasonics, Danbury, CT, USA), respectively. Total protein concentration was determined in duplicate by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and UV–Vis spectrophotometer (8453 UV–Vis Supplies, Agilent Technologies, Waldbronn, Germany) detector using BSA as the protein of reference. For sample centrifugation, a thermostated centrifuge SL16 R (Thermo Fisher Scientific, Langenselbold, Germany) or Mini Spin (Eppendorf AG, Hamburg, Germany) were used as specified for sample treatment. HPLC–ESI–MS/MS analyses were performed on an UltiMate 3000 RSLCnano System (Dionex, Sunnyvale, CA, USA) coupled with an Orbitrap Elite MS detector with ESI or EASY-Spray nanoESI sources (Thermo Fisher Scientific), as specified elsewhere.

A total of 1 x 10⁷ BM-Mϕ were treated with a vehicle control or cytokine for 24 hours and ChIP was performed as previously described [24 (link)]. DNA was fragmented by sonication using a Branson Sonifier 450 (Branson Ultrasonics). For immunoprecipitation, the following antibodies were used: IRF7 (Santa Cruz Biotechnology), STAT1 (Abcam), H3K9me3 (Abcam), Setdb2 (Dr. Yali Dou, University of Michigan), and rabbit polyclonal IgG (Millipore). DNA was assessed by RT-PCR using custom primers (S2 Table).

Bacterial pellets containing induced HIS-S5a protein was thawed and resuspended in a solution containing 50mM Tris pH 8.0 and 10% sucrose. The suspension was incubated with fresh lysozyme to 0.2 mg/l and left on ice for 30 min. The bacteria were lysed by addition of NP40 to a final concentration of 0.1%. Following 10 min of incubation on ice the lysates was sonicated at 30% output two times for 2 min using a Branson Sonifier 450 (Branson Ultrasonics, Danbury, CT), and then centrifuged at 20,000 rpm for 30 min at 4°C in the 70Ti rotor. The supernatant was collected and incubated with Ni-NTA agarose beads (Qiagen Inc., Valencia, CA), for 4 hrs at 4°C with gentle rotation. The mixture was then loaded onto a 10mL column and washed with 3 column volumes of wash buffer (50mM Tris pH 8.0, 350mM NaCl, 10% glycerol, 30mM imidiazole) and 2 column volumes of wash buffer lacking NaCl and then the bound HIS-tagged proteins were eluted with 50mM Tris pH 8.0,10% glycerol, 250mM imidazole. The eluted proteins were dialyzed against 20mM Tris pH 8.0, 150mM NaCl, 10% glycerol buffer and frozen in small aliquots at -80°C.