Rabbit pld2 n term igg

To confirm the presence of endogenous PLD1, PLD2 and S6K proteins in RAW264.7 macrophages, we performed SDS-PAGE and western blot analyses specific for each of these three proteins, as well as using TBP as the equal protein loading control. Approximately 150 μg of protein lysate (lysis buffer composition was: 50 mM HEPES, pH 7.2, 100 μM Na₃VO₄, 0.1% Triton X-100 and 1 mg/ml each of protease inhibitors (aprotinin and leupeptin) was loaded per each lane of the SDS-gels that were then used for Western blot analyses. For western-blot analyses, rabbit PLD1 (F-12) IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (cat. # sc-28314), rabbit PLD2 (N-term) IgG (Abgent, San Diego, CA, USA) (cat. # AP14669a), rabbit S6K IgG (49D7), S6 (cat. # 2217) and pS6 (cat. # 4858). (Cell Signaling Technology, Danvers, MA, USA) (cat. # 2708) and rabbit TBP IgG (Cell Signaling Technology) (cat. # 8515) were utilized as primary antibodies according to the manufacturers’ recommendations. Anti-rabbit or -mouse IgG HRP antibodies were from Cell Signaling Technology (cat. # 7074 and 7076, respectively). Immunoreactivities were detected using enhanced chemiluminescence (ECL) reagents from GE Healthcare (Pittsburgh, PA, USA) (cat. # RPN2106) and autoradiograph film.