Anti pakt 9271

Embryos were fixed in 3.7% paraformaldehyde for 20 min at room temperature, permeabilized with PBS/PVA containing 0.5% Triton X-100 at 37°C for 1 h, and then incubated in PBS/PVA containing 3.0% bovine serum albumin at 37°C for 1 h. Subsequently, the embryos were incubated overnight at 4°C with anti-LC3 (ab58610, 1:100; Abcam, Cambridge, UK), anti-cytochrome C (ab110325, 1:100; Abcam, Cambridge, UK), anti-pAKT (9271, 1:100; Cell Signaling Technology, Danvers, MA, USA), anti-p53 (sc6243, 1:100; Santa Cruz, CA, USA), or anti-OCT4 (sc8628, 1:100; Santa Cruz, CA, USA) antibodies. After washing three times in PBS/PVA, the oocytes and embryos were incubated at 37°C for 1 h with either goat anti-rabbit IgG or rabbit anti-goat IgG. The oocytes and embryos were then stained with Hoechst 33342 for 5 min, washed three times in PBS/PVA, mounted onto slides, and examined using a confocal microscope (Zeiss LSM 710 META, Jena, Germany). Images were processed using Zen software (version 8.0, Zeiss, Jena, Germany).

Ba/F3 was stably transduced with Flag-wild-type and mutated GNB2 (p.G77R) (pMIG vector) as described previously [23 (link)]. Protein levels were evaluated by immunoblot. Cells were lysed, subjected to SDS-PAGE, and transferred to PVDF membrane (Millipore). Each blot was incubated with anti-Flag antibody (#1E6, Wako Pure Chemicals) or anti-Actin antibody (#I-19, Santa Cruz Biotechnology), and its signal was visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore). Phosphorylation profile in the downstream signaling pathway of GNB2 was assessed by immunoblot with anti-total AKT (#9272) and anti-pAKT (#9271) antibodies purchased from Cell Signaling. To investigate an effect of GNB2 on leukemogenesis, Ba/F3 cells transduced with wild-type GNB2, GNB2 mutant, and mock as above, were transplanted in immunodeficient mice. To assess GNB2-associated leukemic involvement, 2 × 10⁶ cells per mouse were intravenously transplanted into NOD/SCID/γc null (NOG) mice (N = 21 in total) after 2.5 Gy irradiation. GFP positivity was measured in bone marrow cells. To further study GNB2-associated tumorigenesis, 1 × 10⁷ cells per experiment were subcutaneously injected into nude mice (N = 30 in total) and the tumor size was measured every seven days.

Embryonic calvarial coronal sutures (E15.5) were lysed in RIPA buffer supplemented with protease inhibitors (Complete Mini; Roche Diagnostics, Indianapolis, IN, USA). Protein extracts (10 µg each) from paired sutures from embryos were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were then incubated with specific primary antibodies for 12 h at 4°C, followed by incubation with horseradish peroxide (HRP)-conjugated secondary antibodies. Protein bands were developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK) and visualized using an LAS 4000 system (Fujifilm, Tokyo, Japan). Primary antibodies, including anti-extracellular signal-regulated kinase (ERK, 9102), anti-phospho (p)-ERK (9101), anti-MAPK kinase (MEK, 9122), anti-p-MEK (91221), anti-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK, 9252), anti-p-SAPK/JNK (9251), anti-p38 (9212), anti-p-p38 (9211), anti-Akt (9272), anti-p-Akt (9271), and anti-Bax (2772), were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Esrp1/2 monoclonal antibodies (201-301-C31S) and anti-β-actin monoclonal antibodies (A1978) were purchased from Rockland (Gilbertsville, PA, USA) and Sigma-Aldrich (Poole, UK), respectively.