P42 44 erk

Neutrophils (1x10⁶, Fig. 1; 0.5x10⁶, fig. S5) prepared at room temperature as described above were resuspended in SDS loading buffer and freeze-thawed and sonicated. Samples were resolved using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 150V and transferred overnight at 35V. For quantification of PI3Kγ subunits, they were immunoblotted with either rabbit α-p101 (Cell Signaling), rabbit α-p84 ((27 (link))), rabbit α-p110γ and p67^phox (Millipore), or mouse α-βcop (a kind gift from N. Ktistakis, Babraham Institute). For p42/44 ERK and PKB activation, bone marrow neutrophils (BMNs) were incubated for 3 min at 37°C prior to fMLP stimulation (or vehicle) for 1 min and stopped with cold PBS. Cell lysates were immunoblotted for P-PKB–Ser⁴⁷³ (Cell Signaling, 1:2500), P-42/44 ERK (Cell Signaling, 1:1000), PKB (Cell Signalling, 1:1000), ERK (Cell Signalling, 1:1000), βcop (gift from N. Ktistakis, 1:250), p47^phox (Upstate, 1:4000) and p67^phox (Millipore, 1:2000). Signal was detected by ECL and bands were quantified by using ImageJ or Image Studio Lite. Where indicated, anti-p67^phox was used as a loading control to normalize for neutrophil input between different bone marrow derived neutrophil preparations.