Purified CD19 B cells from mouse spleens were stimulated with 10 μg/mL lipopolysaccharide (LPS, Sigma, L2880) for 72 h at 5% CO2 and 37 °C. The total protein of the activated B cells was then extracted by RIPA lysis buffer (Beyotime, Shanghai, China, P0013B) containing phosphatase inhibitor (Beyotime, P1050) and protease inhibitor (Beyotime, P1011). The prepared protein was separated on 10% SDS-PAGE gels and transferred on polyvinylidene fluoride membranes (0.45 µm; Millipore, Burlington, MA, USA, IPVH00010). Antibodies specific to Btk (Cell Signaling Technology, Danvers, MA, USA, 8547S), pBtk (Cell Signaling Technology, 87141S), PLCγ2 (Cell Signaling Technology, 34264S), pPLCγ2 (Cell Signaling Technology, 3871S), ERK (Cell Signaling Technology, 4695S), pERK (Cell Signaling Technology, 4370S), NF-κB p65 (Cell Signaling Technology, 4764S), pNF-κB p65 (Cell Signaling Technology, 3033S), and GAPDH (Cell Signaling Technology, 8884S) were incubated with the membranes overnight at 4 °C. The membranes were incubated with secondary peroxidase-conjugated antibodies the next day at room temperature for 1 h. Chemiluminescent signals were detected by the ECL method (Beyotime, Shanghai, China, P0018FS). The relative expression of each band was calculated with Fiji software.
Plcγ2
Manufactured by Cell Signaling Technology
Sourced in United States
PLCγ2 is a protein that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This enzymatic activity is involved in intracellular signal transduction pathways.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (3 mentions)
Phospho p38, by Cell Signaling Technology (3 mentions)
Phospho plcγ2, by Cell Signaling Technology (3 mentions)
P plcγ2, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Nfatc1, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Phospho plcγ2 tyr759, by Cell Signaling Technology (2 mentions)
Phospho tyr1217 plcγ2, by Cell Signaling Technology (2 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
P1050, by Beyotime (1 mentions)
Polyvinylidene fluoride membranes 0.45 m, by Merck Group (1 mentions)
P btk, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
16 protocols using plcγ2
1
Western Blot Analysis of Activated B Cells
2
Nardosinone Modulates Osteoclastogenesis
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Nardosinone (Figure 1A ), purchased from Must Biotechnology (Chengdu, China), was dissolved in dimethyl sulfoxide (DMSO). Alpha modification of Eagle’s minimum essential medium (α-MEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Gaithersburg, MD, United States). Recombinant murine M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, United States). Tartrate-resistant acid phosphatase (TRAP) staining solution, Triton X-100, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, United States). FITC phalloidin was obtained from Yeasen biotech Co., Ltd (Chengdu, China). Primary antibodies targeting GAPDH, IκBα, phospho-Akt, Akt, phospho-ERK1/2, ERK1/2, phospho-JNK1/2, JNK1/2, phospho-p38, p38, phosphor-PLCγ2, PLCγ2, c-Fos and NFATc1 were purchased from Cell Signaling Technology (Danvers, MA, United States). Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kits were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). LPS from P. gingivalis was purchased from InvivoGen (San Diego, CA, United States).
3
Immunoblotting Assay for Cellular Signaling
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Immunoblotting were performed as previously described [32 (link)]. Antibodies including total STAT3, SYK, PLCγ2, AKT, ERK1/2, PARP, caspase 3, and phosphorylated STAT3 (S727), STAT3 (Y705), SYK (Y525/526), PLCγ2 (Y759), PLCγ2 (Y1217), AKT (S473), ERK1/2 (Y204) and RB (S807/811) were purchased from Cell Signaling (Danvers, MA). Cyclin E antibody was purchased from Santa Cruz Biotechnology (Dallas, TX), and β-actin antibody was purchased from Sigma-Aldrich (St Louis, MO). Antibodies used in phosphoflow assay, including phosphorylated STAT3 (S727), SYK (Y525/526), PLCγ2 (Y759), AKT (S473), and ERK1/2 (Y204) were purchased from BD Biosciences (San Jose, CA).
4
Platelet Signaling Pathway Activation
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Collagen, ADP, and thrombin were purchased from Chrono-log Corp. (Havertown, PA, USA). Fura-2/AM and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fibrinogen Alexa Fluor® 488 conjugate was purchased from Molecular Probes (Eugene, OR, USA), and the ATP assay kit was obtained from Biomedical Research Service Center (Buffalo, NY, USA). Antibodies against phospholipase Cγ2 (PLCγ2), phospho-p44/42 (phospho-extracellular signal-regulated kinase (ERK)), p44/42 (ERK), phospho-p38, p38, MEK, phospho-MEK, stress-activated protein kinase (SAPK)/c-Jun N-terminal protein kinase (JNK), phospho-SAPK/JNK, phospho-phosphoinositide 3-kinase (PI3K), PI3K, phospho-Akt, and Akt were acquired from Cell Signaling Technology (Beverly, MA, USA). Ultrapure water was obtained from J. T. Baker (Phillipsburg, NJ, USA). All chemicals were reagent grade.
5
Osteoclast Signaling Pathway Analysis
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Total lysates of human osteoclast precursor cells were prepared from the lysis buffer (Cell Signaling Technology). Total lysates were loaded on 4–12% SDS-PAGE (Bio-Rad), and then western blot analysis was performed using antibodies, pBtk Y223 (Novus Biologicals), pLyn Y396 (Gene Tex), Btk, Lyn, pGab2 Y452, Gab2, pPLCγ2 Y759, PLCγ2, pBLNK, BLNK, NFATc1 (Cell Signaling Technology).
6
Investigating Btk inhibitor-induced apoptosis
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The Btk inhibitors ibrutinib and PLS-123 were synthesized at the laboratory of Dr. Zhengying Pan at Peking University Shenzhen Graduate School according to a previously published procedure [14 (link), 38 (link)]. Antibodies against Caspase-3 (#9662), Caspase-8 (#9746), Caspase-9 (#9508), XIAP (#2045), BCL-xL (#2764), BCL-2 (#2870), MCL-1 (#5453), BAX (#5023), phospho-Tyr223-Btk (#5082), Btk (#8547), phospho-Tyr759-PLCγ2 (#3874), phospho-Tyr1217-PLCγ2 (#3871), PLCγ2 (#3872), phospho-p38 (#9211), p38 (#8690), phospho-Thr308-AKT (#9275), AKT (#9272), phospho-Ser2448-mTOR (#2971), mTOR (#2972), phospho-ERK1/2 (#4370) and ERK1/2(#9102) were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-PARP, phospho-Tyr551-Btk, β-actin (A5441), IgM, Ki-67 antibodies were obtained from BD Biosciences, Sigma (St. Louis, MO, USA) and Abcam (Cambridge, MA).
7
Platelet signaling pathway analysis
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Platelets isolated by gel filtration (2×108/mL) and treated with indicated inhibitors and agonists were pelleted at 960 g (5 min, 37°C). The platelets were then lysed for 30 min on ice with 50 mM Tris-HCl (pH 7.4), 1 % IGEPAL CA-630, 0.25 % sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mM Na3VO4, and 1 mM NaF. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with rabbit anti-human phospho-PLCγ2 Tyr759, phospho-PLCγ2 Tyr1217, phospho-SYK Tyr323, phospho-SYK Tyr525/526 (C87C1), or phospho-Src family Tyr416 (Cell Signaling, Danvers, MA). The membranes were stripped and then probed for normalization with rabbit anti-human SYK (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA) or PLCγ2 (Cell Signaling).
8
Signaling Pathways in Osteoclastogenesis
9
Western Blot Antibody Detection Protocol
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FLAG-tagged PTP1B was detected with anti-FLAG antibody (20543-1-AP [Proteintech]). PTP1B was detected with anti-PTP1B clone FG6-1G (MABS197; Merck Millipore) or with clone H-135 (sc-14021) from Santa Cruz Biotechnology to detect specifically the C-terminal part. Anti-CD22 antibody was purchased from Novus Biological (clone 2H1C4), anti-CD79A (Igα; clone HM47) and anti-SYK (4D10.2) from BioLegend. The antibodies directed against; BTK-pY223, #5082; PLCγ1, #5690; PLCγ2, #3872; PLCγ2-pY759, #3874; SYK-p525/526, #2710; ERK, #4695; ERK-pT202/Y204, #4370; GRB2, #3972; and GAPDH, #2118 were all from Cell Signaling. Anti-phospho-CD22-pY807 (ab32040) antibody was purchased from Abcam. Anti-Myc-tag (66004-1-Ig), anti-APLP2 (15041-1-AP) and anti-CBL (25818-1-AP) were from Proteintech. Anti-BTK (sc-1107) and anti-BCAP (AF4857) antibodies were obtained from Santa Cruz Biotechnology and R&D Systems Inc., respectively. All antibodies were used according to the manufacturer’s instructions. For imaging, a ChemoCam (Intas) equipped with a full-frame 3.2 megapixel Kodak KAF-3200ME camera was used. Western blot signals were quantified with Quantity One 4.6.9 (Bio-Rad). For statistical analysis, two-sample t tests were performed over three replicates using Microsoft Excel 2013. Error bars represent the SEM.
10
Activation of STAT Signaling Pathways
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