35s methionine

In vitro transcribed RNAs were translated in 10 μl of the supplemented untreated RRL 50% (v/v) (Promega Co., Madison, WI, USA) in the presence of KCl (75 mM), MgCl₂ (0.5 mM), 20 μM of amino acids mix minus methionine and 0.6 μCi of [³⁵S]-methionine (GE Healthcare Life Sciences) for 30 min at 30°C. Reactions were stopped with 2× SDS-loading buffer and the products were resolved by 15% SDS–PAGE. Gels were dried and subjected to autoradiography using Biomax films (Eastman Kodak Co.).

Platelets (1 × 10⁹ total) were placed in 1 mL of methionine-free M199 medium for 2 h followed by the addition of 50 µCi (1 Ci = 37 GBq) of [³⁵S]methionine (GE Healthcare, Wauwatosa, WI, USA)) for each sample. After the addition of labeled methionine, the platelets were left in solution without any activator for 30 min. Then they were activated with thrombin, PAF or oxidized phospholipids in the presence of fibrinogen or not, or left quiescent without any activator (= control). Platelets were gently rocked and six hours later, the platelets were washed with Tris-buffer, the suspensions were centrifugated and the supernatants were removed. The cell pellets were lysed in RIPA-buffer on ice and identical volumes of the platelet lysates were separated by SDS-gel electrophoresis on a 9% gel. The gels were dried for 2 h in an 80 °C vacuum oven and exposed overnight to Kodak MS film placed in a −80 °C refrigerator.