Js13.1 rotor

Nicotiana tabacum (L.) pollen collected in the Botanical Garden Città Studi, as described above, was hydrated in a humid chamber overnight. Pollen (3 mg/mL) was germinated in BK medium as reported above, with or without squalestatin 1 μM or myriocin 5 μM. Pollen tubes were rinsed with 10 mL of incomplete TNE buffer (mM Tris, 150 mM NaCl, mM EGTA, 1 mM PMSF, 10 μg/mL TAME) containing 12% sucrose, with or without squalestatin 1 μM or myriocin 5 μM and centrifuged at 2000 r.p.m. for 10 min at 10 °C in a Beckmann JS13.1 rotor. Pollen tubes were homogenized on ice in two volumes of complete TNE (mM Tris, 150 mM NaCl, mM EGTA, 1 mM PMSF, 10 μg/mL TAME, 10 μg/mL leupeptin, 10 μg/mL pepstatin A, 4 μM aprotinin, 8 μM antipain) using a 2 mL Potter (teflon/glass) homogenizer. The homogenate was centrifuged at 572 g for 4 min at 4 °C and the post nuclear supernatant loaded onto a 20% sucrose cushion (3 mL) in incomplete TNE buffer and centrifuged at 64,200 g (Beckman SW-60 rotor) for 30 min at 4 °C. The P2 pellet was resuspended in cold, complete PEM buffer. Aliquots of P2 and supernatant (S2) were protein-assayed (Bradford) using BSA as standard protein.

Mammary gland pieces were prepared with the use of scissors as above and washed 3 times for 10 minutes in 0.25 M sucrose at 4°C to remove milk constituents, and further minced using a homemade multi-mounted razor blade device. All subsequent steps were performed at 4°C. Tissue was homogenized in a 20 ml Teflon-glass homogenizer (BB, Thomas scientific) for 3 strokes. The homogenate was filtered through a piece of 150 µm polypropylene mesh (ZBF, Rüschlikon, Switzerland) and centrifuged at 8700 g in a Beckman JS 13.1 rotor for 13 minutes. The resulting supernatant is referred to as PNS. Membrane-bound organelles were sedimented from the PNS by centrifugation at 110,000 g for 1 hour. Total rough ER microsomes were prepared from the PNS by differential centrifugation followed by sucrose density gradient, as described by Paiement et al. for liver tissue [19] , with the minor modifications reported in Le Parc et al. [15] (link). The final rough ER microsomal pellet was resuspended in 2 ml of 2 mM imidazole pH 7.4, 0.25 M sucrose, and aliquots were stored at −80°C. The rough ER microsomal fraction was previously characterized [15] (link). Protein concentration in the fractions was determined.

Tobacco pollen tubes were grown in liquid BK medium for 2 h and then rinsed with 10 ml HEM buffer pH 7.4 (25 mM HEPES, 2 mM EGTA, 2 mM MgCl₂, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg ml⁻¹ TAME, 10 µg ml⁻¹ leupeptin, 10 µg ml⁻¹ pepstatin A, 4 µM aprotinin, 8 µM antipain) containing 12% sucrose. After centrifuging at 2000 r.p.m. for 10 min at 10°C in a Beckmann JS13.1 rotor, pollen tubes were homogenized on ice in two volumes of HEM buffer containing 10% mannitol. The homogenate was centrifuged at 572g for 4 min at 4°C and the supernatant loaded onto a 0.5 M sucrose cushion (3 ml) in HEM buffer and centrifuged at 64 200g for 23 min at 4°C. The pellet containing microsomes (P2) was resuspended in PEM buffer. Aliquots of P2 and supernatant (S2) were protein assayed (Bradford method) using BSA as standard protein. For peripheral protein stripping, P2 was incubated with 0.8 M KCl in HEM buffer, loaded onto a 0.5 M sucrose cushion in HEM buffer and centrifuged at 64 200g for 23 min at 4°C. These organelles, resuspended in HEM buffer, were used for binding experiments.