Type 50.2 ti rotor

Membranes harvested from discontinuous sucrose gradients were diluted in 20 mM HEPES, pH 7.5 and pelleted at 45,000 rpm (180,000 ×g) for 2.5 h using a Beckman Type 50.2 Ti rotor at 4 °C. Pelleted membranes were resuspended in approximately 100–200 μl of 20 mM HEPES, pH 7.5 and the absorbance spectrum used to record the OD₈₇₅ value. 7.5 OD₈₇₅ units of resuspended membranes, equivalent to ~ 43 nmol of BChl, were solubilised in 3% β-dodecyl maltoside (β-DDM) in a total volume of 250 μl before 1 h of centrifugation at 15,000 rpm at 4 °C in a refrigerated microcentrifuge. The supernatant was collected and layered on top of a discontinuous sucrose gradient containing 20%, 21.3%, 22.5%, 23.8% and 25% sucrose, 20 mM HEPES and 0.03% β-DDM. Gradients were centrifuged in a Beckman SW41 Ti rotor at 27,000 rpm (90,000 ×g) for 40 h. Digital photos of the gradients were taken and pigmented bands were harvested for downstream processing. The dimer:monomer ratios were estimated using ImageJ software (NIH, Bethesda, USA) to analyse densitometry of photographs of the gradients, as described in [22] (link).

Membranes from 2 to 4 l of photosynthetically grown cells were harvested from discontinuous sucrose gradients, diluted in 20 mM HEPES, pH 7.5 and pelleted at 45 000 rpm (180000 × g) for 2.5 h using a Beckman Type 50.2 Ti rotor at 4°C. Membranes were resuspended to an OD₈₇₅ of 30 and incubated in 0.1–3.0% β‐DDM for 30 min at 4°C. Insoluble material was removed by centrifugation at 20 000 rpm (32 000 × g) in a Beckman JA‐25.50 rotor at 4°C. The supernatant was loaded onto a pre‐equilibrated 150 μl anti‐FLAG M2 affinity resin column. The column was washed with 15 volumes of wash buffer containing 0.04% β‐DDM. FLAG‐tagged and associated proteins were desorbed by the addition to the plugged column of 100 μg FLAG peptide dissolved in 500 μl wash buffer. The resin was transferred to a cryovial and rotated for 1 h at room temperature prior to a 1500 × g centrifugation for 5 min in a Costar Spin‐X centrifuge tube containing a cellulose acetate membrane with 0.22 μm pores to separate proteins from the resin.

Normal and SILAC-labeled yeast cells were collected by centrifugation at 4500 rpm for 10 min at 4°C, washed with cold deionized water and resuspended in 20 ml of lysis buffer (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 5 mM MgCl₂, 1 mM DTE) containing protease inhibitors (1 mM PMSF, 2 µg/ml aprotinin, 5 µg/ml pepstatin A, 1 µg/ml leupeptin). Cells were lysed with 5 to 6 cycles in French Press (Thermo Electron Corporation) at 18000 psi. Cell lysate was centrifuged twice at 15500 rpm for 15 min at 4°C in Type 50.2 Ti rotor (Beckmann). The supernatant was collected and the ribosomal particles were pelleted by centrifugation through a 20% sucrose cushion in buffer A (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM MgCl₂, 1 mM DTE) at 35000 rpm for 24 h (ω²t = 1.16*10¹²) at 4°C in in the same rotor. The pellet was resuspended in 2 ml of buffer A at 4°C for at least 2 hours, cleared at 15000 rpm for 20 min at 4°C in the same rotor and absorbance at 260 nm (A₂₆₀) was measured. Thirty-five to forty A₂₆₀ units (U) of ribosomal particles was carried onto a 10%–30% (w/w) sucrose gradient in buffer A and centrifuged at 20500 rpm for 16 h 15 min (ω²t = 2.7*10¹¹) at 4°C in SW28 rotor (Beckmann). Fractions containing 80S ribosomal particles were collected, diluted 2 times with buffer A and concentrated using 100 000 Da MwCo filters (Millipore). Ribosomes were frozen in liquid nitrogen and stored at –80°C.

Lyophilized rabbit skeleton muscle acetone powder was rehydrated and ground in G-buffer (5 mM Tris-HCl, pH 8, 0.2 mM ATP, 0.1 mM CaCl₂, 0.5 mM DTT, annd 0.1 mM sodium azide), and then cleared by centrifugation at 27,000 × g in the JA25.50 rotor (Beckman Coulter, Inc., USA). Solubilized actin in the supernatant was collected and polymerized by adding 50 mM KCl and 2 mM MgCl₂ for 1 h, followed by the addition of 0.8 M KCl for 30 min at 4 °C. F-actin was pelleted by centrifugation at 147,600 × g using a Type50.2Ti rotor (Beckman Coulter, Inc., USA) and then depolymerized by brief sonication and dialysis against G-buffer for 48 h at 4 °C. Monomeric Ca²⁺-ATP-actin was cleaned by centrifugation at 193,900 × g for 2.5 h using the SW 55Ti rotor (Beckman Coulter, Inc., USA). The supernatant was collected and loaded to Sephacryl S-300 HR for gel filtration chromatography using G-buffer (5 mM Tris-HCl, pH 8, 0.2 mM ATP, 0.1 mM CaCl₂, 0.5 mM DTT, and 0.1 mM sodium azide) to obtain monomeric Ca²⁺-ATP-actin.

H69 cells, immortalized human normal cholangiocytes (a gift from Dr. G. J. Gores, Mayo Clinic, MN), were maintained as described^{36 (link), 37 (link)}. Our immortalized small and large cholangiocytes were also maintained as described^{7 (link), 11 (link), 14 (link), 38 (link)}. Stable transfected knockdown of SCT or SR in murine large cholangiocyte lines using short hairpin RNA (shRNA) plasmids were established in our laboratory and maintained as described^{7 (link), 11 (link)}. Human primary hepatocytes and culture media for these cells were purchased from ScienCell Research laboratories (Carlsbad, CA) and maintained according to the manufacturer’s instruction. Cells (3 × 10⁵) were cultured in T175 flasks containing 30 mL of culture media with 1× phosphate buffered saline (PBS), LPS (50 ng/mL, Sigma-Aldrich, St Louis, MO), or SCT (100 nM, Bachem, Bubendorf, Switzerland) for 72 hours. Supplement fetal bovine serum (FBS) was depleted of EVs by ultracentrifugation at 120,000 × g for 18 hours (Type 50.2 Ti rotor, Beckman Coulter, Brea, CA)³⁹ . All cells were cultured at 37 °C and 5% CO₂.