Anti phospho plcγ2

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).

Methanol extract from the S. hexaphylla leaf was purchased from the Korean Plant Extract Bank (Daejeon, Korea). Recombinant soluble human M-CSF and RANKL were obtained from PeproTech EC Ltd. (London, UK). Monoclonal β-actin antibody was obtained from Sigma (St. Louis, MO, USA). Anti-Akt, anti-phospho-Akt, anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-phospho-IκB, and anti-phospho-PLCγ2 were purchased from Cell signaling Technology Inc. (Beverly, MA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fetal bovine serum (FBS), α-minimum essential medium (α-MEM), and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). All other chemicals were of analytical grade or complied with the standards required for cell culture experiments. Cyclohexamide (CHX), MG132, and Ac-Leu-Leu-nle-h (ALLN) were obtained from Calbiochem (San Diego, CA, USA).

CD95L treated (40 ng/ml) or non-treated cells were washed with PBS containing phosphatase inhibitors, pelleted, and lysed on ice for 30 min with Pierce IP Lysis buffer (Fisher Scientific, Germany) containing vanadate, inhibitors for phosphatase and proteinase. Lysates of 500 μg protein were immunoprecipitated at 4°C for 4 hr with the anti-mouse CD11a (M17/4, biolegend), anti-mouse Btk (Cell Signaling, USA), anti-mouse CD11b (M1/70, ebioscience, USA) antibodies or the corresponding isotype controls. Afterward, 40 μl Dynabeads M-280 Streptavidin was added to each sample and incubated for 1 hr at 4°C with rotation. Beads were washed 5 times with 1 ml of lysis buffer. The immunoprecipitates were released by cooking the beads with 40 μl of 2x laemmli buffer at 95°C for 5 min.
Immunoblotting was performed as previously described (Letellier et al., 2010 (link)). Membranes were probed with following antibodies respectively: anti-phospho-Syk (Tyr319, 352), anti-phospho-Btk (Tyr223), anti-phospho-PLCγ2 (Tyr1217), anti-Syk, anti-Btk, anti-PLCγ2 (Cell Signaling, USA), anti-Rap1 (Fisher Scientific), anti-mouse CD11a, anti-CD95 (M20, Santa Cruz Biotechnology, Germany), anti-mouse CD11b (Novus Biologicals, USA). Western blots were quantified with ImageJ software and normalized to the respective loading controls.