Pierce ecl2

BMDCs were harvested, washed in cold PBS, and lysed in 50mM Tris-HCl (pH 7.9), 300mM NaCl, 1% Triton X-100, supplemented with protease inhibitor and phosphatase inhibitor cocktails (respectively Complete Protease and Phostop, Roche). Whole cell extracts were cleared by centrifugation at 14,000 rpm for 10 min at 4°C. Protein concentrations were determined using the Bradford method. Samples were denatured in Laemmli buffer for SDS-PAGE resolution. Proteins were transferred onto a PVDF membrane (Millipore). Membranes were blocked with 7% evaporated milk in PBS 0.2% Tween and were incubated with primary antibodies and peroxidase-conjugated secondary antibodies (all diluted in PBS 7% milk 0.2% Tween). Bound antibodies visualized using Amersham™ ECL or Pierce® ECL2, and imaged using developing machine or Amersham™ Imager 600 (GE Healthcare). Anti-Sec22b was from Santa Cruz (sc-101267), anti-β-Actin was from Cell Signaling (3700).

Macrophages were washed in cold PBS, then lysed in 50mM Tris-HCl (pH 7,9), 300mM NaCl, 1% Triton X-100, supplemented with protease and phosphatase inhibitor cocktails (respectively Complete Protease and Phostop, Roche). WCE were centrifuged at 3,640×g, 10 min at 4°C. Protein concentrations were determined using the Bradford method. Samples were denatured in Laemmli sample buffer for SDS-PAGE resolution. Proteins were transferred onto a PVDF membrane (Millipore). Membranes were blocked with 7% evaporated milk in PBS 0.2% Tween and incubated with primary antibodies and peroxidase-conjugated secondary antibodies (all diluted in PBS 7% milk 0.2% Tween). Bound antibodies were visualized using the Amersham^™ ECL or Pierce® ECL2 detection reagents, and imaged using developing machine or Amersham^™ Imager 600 (GE Healthcare). All immunoblots represent at least 3 independent experiments. All blots were probed for β-Actin as a loading control.