Antibodies against phospho akt

βBA, obtained from Sigma-Aldrich (St. Louis, MO, USA), was dissolved and prepared with 5, 10, 20, and 30 mM stock solutions in DMSO. The control group was added 0.1% DMSO. TRIzol reagent was obtained from Life Technologies (Carlsbad, CA, USA). A monoclonal anti-β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against phospho-Akt, anti-Akt, anti-phospho-p38, anti-total p38, anti-phospho-IκB, anti-Bruton’s tyrosine kinase, and anti-phospho-PLCγ2 were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-phospho-Btk antibody was obtained from GeneTex (Irvine, CA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Donkey anti-rabbit and anti-mouse immunoglobulin secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Immunoblotting were performed as described previously [9 (link), 10 (link)]. Briefly, protein lysates from ES cells were isolated with lysis buffer (25 mM HEPES, 10 mM Na₄P₂O₇・10H₂O, 100 mM NaF, 5 mM EDTA, 2 mM Na₃VO₄, 1% Triton X-100). Protein lysates were subjected to SDS-PAGE and Western blotting. The blots were probed with the indicated antibodies and their respective HRP-conjugated secondary antibodies obtained from JacksonImmuno Research Laboratories (West Grove, PA). Antibody against Hsp72 was purchased from Stressgen Biotechnologies (Victoria, BC, Canada). Antibodies against phospho-Akt, Akt, phospho-extracellular signal-regulated kinase (ERK), ERK, phospho-p38, p38, phospho-c-jun NH₂-terminal kinase (JNK), and JNK were purchased from Cell Signaling Technology (Beverly, MA). Densitometric analysis was performed by ImageGauge software.