Anti pld1

Cell cultures were washed with ice-cold PBS, lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM EDTA, 4 mM NaF, 0.5% NP-40, 1 mM PMSF) and centrifuged at12,000 × g at 4 °C for 5 min to extract the protein in the supernatant. The concentration of protein was measured with a BCA assay kit (Pierce). Equal amounts of samples were denatured and subjected to SDS-PAGE. After separation, proteins were transferred to nitrocellulose membranes (Pall). The membranes were blocked with 5% nonfat milk in TBST (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20) for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. After washing with TBST three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma Aldrich & Origene) overnight at 4 °C, washed again and finally were developed with ECL solutions (Santa Cruz Biotechnology & Millipore). The immunoreactive bands were scanned and analyzed quantitatively by densitometry with Quantity One (Bio-Rad). The primary antibodies used in Western blots were rabbit polyclonal anti-PLD1 (Cell Signaling Technology), mouse monoclonal anti-N-cadherin 3B9 (Invitrogen), rabbit monoclonal anti-ADAM10 (Abcam), mouse monoclonal anti-human TfR (Invitrogen) and mouse monoclonal anti-β-actin (TA-09; Origene).

Samples were separated on 10% sodium dodecyl sulfate (SDS)-Laemmli gels and transferred by electroblotting onto nitrocellulose membranes (Biostep). Membranes were blocked in 0.1% Tween/TBS-buffer with 5% dry milk and incubated with antibodies detecting phosphorylated/non-phosphorylated proteins. We used anti-pT183/Y185-JNK-1/2/JNK-1/2, anti-pS176/177-IKK1/2/IKK1/2, anti-pT345/S346-IRAK4, anti-pS32-IκBα/IκBα, anti-PLD1 and anti-tubulin (Cell Signaling; except anti-IKK1/2 from Santa Cruz and anti-tubulin from Sigma). Membranes were washed in 0.1% Tween/TBS and incubated with HRP-conjugated secondary antibodies: anti-rabbit-Ig, anti-goat-Ig (Santa Cruz) and anti-mouse-Ig (Thermo). Detection was performed using ECL reagent (Pierce).