Phospho plcγ1

Formalin-fixed, paraffin-embedded tissue specimens from 38 cases of AITL were prepared as 4-μm-thick slides by the Pathology Department for IHC. Antibodies against phospho-ZAP70 (Tyr 493, 1:100, LifeSpan BioSciences, Seattle, WA, USA), phospho-ITK (Tyr512, 1:50, LifeSpan BioSciences, Seattle, WA, USA), and phospho-PLCγ1 (Tyr783, 1:500, Cell Signaling Technology, Danvers, CA, USA) were used for immunohistochemical analysis. The immunohistochemical assay was performed by the BenchMark XT automated slide processing system (Roche, Mannheim, Germany). Each sample was graded independently by two pathologists from the department of pathology in Peking University Cancer Hospital & Institute.
The expression of the phosphoprotein was semi quantitatively estimated as the immunostaining scores. The percentage of density displayed the fraction of positive staining tumor cells (0–100%). The intensity score represented the staining intensity (‘0’ as negative, ‘1’ as weak positive, ‘2’ as moderate positive and ‘3’ as the strong positive). Slides were visualized through an Olympus BX51 microscope (Olympus, Melville, NY, USA) and photographed with Leica DM6000B camera. The images were analyzed with Leica Aperio Versa 200 (Leica Camera AG, Somme, Germany). Tumors were considered positive for phosphoprotein when ≥ 10% of the neoplastic cells demonstrated phosphoprotein staining.

Nuclear and cytoplasmic proteins were extracted from RBL-2H3 cells. Protein concentration was determined by BCA as previously described [13 (link)], and 20 μg of protein was subjected to SDS-PAGE analysis. After 1 h blocking, different primary antibodies (1 : 1000) were added and incubated overnight at 4°C, and different secondary antibodies were incubated for 1 h at 37°C. Antibodies including NF-κBp65 (#8242), phospho-NF-κBp65 (#3039), PARP (#9532), IκBα (#4814), phospho-IκBα (#2859), p38MAPK (#8690), phospho-p38MAPK (#4511), ERK (#4695), phospho-ERK (#4370), JNK (#9258), phospho-JNK (#9255), Syk (#13198), phospho-Syk (#2710), Lyn (#2796), phospho-Lyn (#2731), Gab2 (#3239), phospho-Gab2 (#3881), PLC-γ1 (Tyr, #2821), phospho-PLCγ1 (#8713), HMGB1 (#3935), TLR4 (#14358), MyD88 (#3699), Nrf2 (#12721), HO-1 (#82206), Keap1 (#8047), β-actin (#3700), anti-rabbit IgG(H + L) (#14708), and anti-mouse IgG (H + L) (#14709) were all purchased from Cell Signaling Technology (Beverly, MA, USA). Protein bands were analyzed using an optical density scanner with a Gel Doc XR system (Bio-Rad, USA).

MEM-α medium, 1 × DPBS, penicillin, streptomycin and fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences (Hyclone™, Logan, UT, USA). The EZ-Cytox cell viability assay kit was obtained from Daeillab Service Co. (Seoul, Korea). Specific antibodies against phospho-cPLA₂, phospho-ERK, phospho-JNK, phospho-LYN, phospho-p38, phospho-PKCδ, phospho-PLCγ1, phospho-SYK and COX-2 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). A specific antibody against phospho-FYN was obtained from Biorbyt Ltd. (Cambridge, UK). A specific antibody against α-tubulin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). An enzyme immunoassay (EIA) kit for PGD₂ was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, and IL-6 were purchased from e-Bioscience, Inc. (San Diego, CA, USA). 4-Nitrophenyl-N-acetyl-β-d-glucosaminide (p-NAG), dinitrophenyl-human serum albumin (DNP-HSA) and DNP-immunoglobulin E (DNP-IgE) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.

Cells were stimulated with antibody-coated goat-anti-mouse Dynabeads (Invitrogen) at the ratio of 4 beads per cell. Antibodies used for bead coating include Myc-tag antibody (9B11, mouse IgG2a, Cell Signaling), NKG2A antibody (Z199, mouse IgG2b, Beckman Coulter), control mouse IgG2b (MOPC-141, Sigma), and control mouse IgG2a (UPC-10, Sigma). 10⁷ beads were coated for 30 min at 37°C with 2 μg total IgG at different combinations: 1 μg mIgG2a + 1 μg mIgG2b, 1 μg mIgG2a + 1 μg anti-NKG2A, 1 μg anti-Myc-tag + 1 μg mIgG2b, or 1 μg anti-Myc-tag + 1 μg anti-NKG2A. Beads were washed with hank’s-balanced-salt-solution (HBSS, Corning) in 1% FCS to remove excessive antibodies and resuspended in the same buffer. Pre-chilled beads and cells were mixed on ice and incubated in a 37°C water bath for 10 min. Cells were washed and lysed before analyzing by western blot using phospho-Erk1/2 (Cell Signaling #9101) and phospho-PLCγ1 (Cell Signaling #2821) antibodies.

For experiments described in Fig 3E, mature OT‐1 CTLs were pretreated with 100 nM of MB. These cells were then seeded in 6‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media supplemented with 20 μg/ml of mPD‐L1 protein and 100 nM of MB for 2 min. Cells were harvested for preparing protein lysate using NP40 lysis buffer. Equal fractions of the lysates were subjected to SDS–PAGE and blotted with Phospho‐Zap‐70 (1:1,000, Cell Signaling Technology, 2717), ZAP70 (1:1,000, Cell Signaling Technology, 3165), Phospho‐PKCθ (1:1,000, Cell Signaling Technology, 9377), PKCθ (1:1,000, Cell Signaling Technology, 13643), Phospho‐PLCγ1(1:1,000, Cell Signaling Technology, 14008), PLCγ1(Cell Signaling Technology, 5690), Phospho‐AKT(1:1,000, Cell Signaling Technology, 4060S) and AKT (1:1,000, Cell Signaling Technology 9272S).