Phospho plcγ

Homogenized liver and spleen tissues, and splenocytes from spleen of WT mice and Jurkat T cells lysed by protein extraction solution (PRO-PREP, iNtRON Biotechnology) containing protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Total proteins (30 μg) were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk overnight and then incubated with primary antibodies (diluted 1 : 1000) for 1 h at room temperature. The membranes were immunoblotted with following primary antibodies: Phospho-STAT1, Phospho-JAK3, Phospho-JNK, Phospho-PLCγ, Phospho-IκB (Cell Signaling Technology, Beverly, MA, USA), STAT1, JAK3, JNK, PLCγ, IκB (Santa Cruz Biotechnology, Dallas, TX, USA). After washing with Tris-buffered saline containing 0.05% Tween-20 (TBST), the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1 : 3000) for 1 h at room temperature. Binding of antibodies to the PVDF membrane was detected with enhanced chemiluminescence solution (Amersham Bioscience, Buckinghamshire, UK) and X-ray film (AGFA, Mortsel, Belgium).

Embryos were dissected at E11.5 and fixed in 4% paraformaldehyde solution (PFA) (pH 7.4) at 4°C overnight. After the tissues were soaked in PBS/1% Triton X-100 for 20 min and rinsed with PBS several times, they were blocked with PBS (0.3% Triton X-100, 5% normal donkey serum and 1x PBS) overnight at 4°C. The tissues were incubated overnight at 4°C with primary antibodies against phospho-ERK (Cell Signaling Technology, 4,370; 1:100), phospho-AKT (Cell Signaling Technology, 8,272; 1:100), and phospho-PLCγ (Cell Signaling Technology, 8,713; 1:100). The tissues were washed 3 times with PBS for 30 min each, incubated with Cy5-conjugated donkey anti-rabbit secondary antibody (Jackson, 1:500) overnight at 4°C and washed several times with PBS. Images of stained tissue were captured using a Zeiss-LSM880 with Airyscan confocal laser scanning microscope and ZEN ImageJ software (Zeiss, Germany). The details about how the measurements were conducted on the confocal images are described in the literature (25 (link)). ImageJ was used to quantify the amount of fluorescence as mean gray value of pERK, pAKT, PLCγ and compare the results among four groups. ImageJ was also used to determine the number of stained cells by phosphorylation of histone H3 (PHH3) to compare the results among these groups.

Eagle’s Minimum Essential Medium (EMEM) and other cell culture supplies, including fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were obtained from ThermoFisher Scientific (Waltham, MA, USA) or Lonza (Walkersville, MD, USA). Both mouse monoclonal DNP antibody and DNP-BSA were provided by Sigma-Aldrich Chemicals (St. Louis, MO, USA). All antibodies against total Syk, phospho-Syk, phospho-PKCμ, phospho-PLCγ, total ERK, phospho-ERK, total cPLA2, phospho-cPLA2 and COX2 were purchased from either Cell Signaling Technology (Boston, MA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).