For detection of phosphorylation of each signaling molecule, cells were washed twice with 1 mM ice-cold sodium orthovanadate (Sigma-Aldrich) in PBS, then lysed with CelLytic M (Sigma-Aldrich) supplemented with a 1% protease inhibitor cocktail (Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein from each sample (10 μg) was separated using 4–12% sodium dodecyl sulfate-polyacrylamide gel (NuPAGE: Invitrogen) and analyzed by western blotting. Blotted membranes were incubated with antibodies for cyclin D1 (1:500, Cell Signaling Technology), Sox2 (1:500, Abcam), P-Akt (1:500, Cell Signaling Technology), Akt (1:500, Cell Signaling Technology), PI3K (1:500, Cell Signaling Technology), P-PLCγ (1:500, Cell Signaling Technology), P-Stat5 (1:500, Cell Signaling Technology), P-Erk 1/2 (1:500, Cell Signaling Technology), Erk 1/2 (1:500, Cell Signaling Technology), and Gapdh (1:500, Cell Signaling Technology), then signals were detected with an ECL kit (Amersham Biosciences) and visualized using an Image Quant LAS 4000 system (GE Healthcare, Life Science).
P plcγ
Manufactured by Cell Signaling Technology
Sourced in United States
P-PLCγ is a phospholipase C enzyme that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate the secondary messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (6 mentions)
P syk, by Cell Signaling Technology (5 mentions)
P erk1 2, by Cell Signaling Technology (4 mentions)
Erk1 2, by Cell Signaling Technology (4 mentions)
P erk, by Cell Signaling Technology (4 mentions)
P jnk, by Cell Signaling Technology (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Dnp bsa, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Raw 264.7 atcc tib 71, by American Type Culture Collection (2 mentions)
Rbl 2h3, by American Type Culture Collection (2 mentions)
P lyn, by Cell Signaling Technology (2 mentions)
4 nitrophenyl n acetyl β d glucosaminide p nag, by Merck Group (2 mentions)
Monoclonal anti dnp ige, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Biowest (2 mentions)
β actin antibody, by Merck Group (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Dectin 1, by Abcam (2 mentions)
20 protocols using p plcγ
1
Western Blot Analysis of Signaling Molecules
2
Mesothelin CAR-T Cell Signaling Dynamics
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Example 7
Human normal donor anti-mesothelin CAR-T cells were stimulated with mesothelin-Fc coated Dynabeads at a 3:1 beads to T cell ratio at 37° C. for 5 or 10 minutes. Protein lysates were collected and quantified by Bradford Assay and electrophoresed on a polyacrylamide gel. Protein lysates were immunoblotted with antibodies for pAKT, AKT, pPLCγ, PLCγ, pERK, ERK (Cell Signaling Technologies), pVav (Abcam), and Vav (Santa Cruz Biotechnology) and exposed through chemiluminescence.
Introducing the YMFM (SEQ ID NO: 1937) mutation in the CD28 costimulatory domain led to altered signaling in T cells, reducing phosphorylation of Vav, PLCγ, and ERK (
3
Western Blot Analysis of Aortic Proteins
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Aortic tissue homogenates were dissolved in sample buffer, run on a NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen, CA, United States), and transferred to nitrocellulose membranes using the iBlot transfer system (Invitrogen). Membranes were washed in PBS and blocked for 1 hr at room temperature with 5% instant non-fat dry milk dissolved in PBS containing 1% Tween-20 (Sigma, MO, United States) (PBS-T). Equal protein loading of samples was determined by a protein assay (Bio-Rad, CA, United States) and confirmed by probing with antibodies against β-Actin (Sigma). Membranes were probed overnight at 4° centigrade with primary antibodies against pSmad3 (1880-1; Millipore), Smad3 (#9513; Cell Signaling), pERK1/2 (#4370; Cell Signaling), ERK1/2 (#4695; Cell Signaling), pPKCβ (#75837; Abcam, United Kingdom), PKCβ (#32026; Abcam), pPLCγ (#2821; Cell Signaling), and PLCγ (#2822; Cell Signaling), dissolved in PBS-T containing 5% milk. Blots were then washed in PBS-T and probed with HRP-conjugated anti-rabbit secondary antibody (GE Healthcare, United Kingdom) dissolved in PBS-T containing 5% milk at room temperature. Blots were then washed in PBS-T, developed using SuperSignalWest HRP substrate (Pierce Scientific, IL, United States), exposed to BioMax Scientific Imaging Film (Sigma), and quantified using ImageJ analysis software (NIH, MD, United States).
4
Protein Expression Analysis in PDAC
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Lysates were diluted with 4× NuPage buffer and run on Invitrogen NuPage 4 to 12% Bis-Tris gels. The antibodies employed were KRAS (Sigma; 3B10-2F2), UGP2 (Santa Cruz Biotechnology; sc-377089), YAP (Cell Signaling; 14074), EGFR (Cell Signaling; 4267), p-PLCγ (Cell Signaling; 3233), p-GAB1 (Cell Signaling; 3234), and B-ACTIN (Sigma; A5441). Immunohistochemistry of xenografts was performed by HistoWiz using antibodies against EGFR (EP38Y) and UGP2 (Santa Cruz Biotechnology; sc-377089; 1:200). Immunohistochemistry of historic deidentified PDAC samples used antibodies against YAP1 (Cell Signaling; 14074) and UGP2 (Santa Cruz Biotechnology; sc-377089). To analyze tissue microarray staining, blinded data extraction for mean intensity and percent area was conducted using inForm software from PerkinElmer. Normalization and χ2 statistical analysis were conducted using Matlab.
5
In Vitro Cell Culture Protocols
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Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Biowest (Kansas City, MO, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS, 4-nitrophenyl n-acetyl-b-d-glucosaminide (p-NAG), and monoclonal anti-DNP-IgE were supplied by Sigma–Aldrich (St. Louis, MO, USA). DNP-BSA was procured from Invitrogen (Gaithersburg, MD, USA). Primary antibodies against p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-Lyn, Lyn, p-Syk, Syk, p-PLCγ, PLCγ, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA), and FcεRIγ, from LSBio (Seattle, WA, USA). SensiFAST SYBR No-ROX kit mix was purchased from Biolines (Seoul, Korea), and human filaggrin, AQP3, and HA ELISA kits were obtained from CUSABIO (Seoul, Korea). Rat basophilic leukemia (RBL-2H3, ATCC® CRL-2256) and murine macrophage (RAW264.7, ATCC® TIB-71) cells were procured from the American Type Culture Collection (ATCC), while human immortalized keratinocyte (HaCaT) cells were obtained from Prof. Lee of Chosun University in Korea.
6
Investigating Ang1-Mediated Signaling Pathways
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APN, Ang1, PLCγ and PKCα antibodies were purchased from Santa Cruz Biotechnology (CA, USA). p-PLCγ and p-PKCα antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). All siRNAs (ON-TARGETplus) were obtained from Dharmacon Research (Lafayette, CO, USA). Taqman® one-step PCR Master Mix, qPCR primers and probes were obtained from Applied Biosystems (Foster City, CA, USA). β-Actin antibody and pharmacological inhibitors were obtained from Sigma-Aldrich (St. Louis, MO, USA).
7
Western Blot Signaling Pathway Analysis
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Cells were harvested and lysed using RIPA buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 2.5 mM NaF, 5 mM Na3VO4, and protease inhibitor cocktail tablet (Roche, # 11–878-580–001). Proteins were separated and transferred to an NC membrane. The membranes were blocked using 5% skim milk in TBS-T buffer. The p-c-KIT (Tyr703, # 3073), p-AKT (Ser473, # 9271), p-ERK (Thr202/Tyr204, # 8544), ERK (# 4695), p-PLCγ (Tyr783, # 14008), p-STAT3 (Tyr705, # 4074), and PARP (# 9542) antibodies were obtained from Cell Signaling Technologies. The β-actin (# sc-47778) and STAT3 (# sc-482) antibodies were obtained from Santa Cruz Biotechnology. AKT (A18120) antibody was purchased from ABclonal. Each primary antibody was incubated overnight at 4 °C. The secondary antibody (GenDEPOT) was incubated for 1 h at room temperature. Proteins were detected using ECL substrate.
8
Protein Extraction and Western Blotting
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For protein extraction, tissues were homogenized in ice-cold RIPA buffer. Total protein was quantified using the DC Protein Assay according to the manufacturer’s instructions (BioRad, Hercules, CA). Western blotting was performed as previously described with minor modifications13 (link). Briefly, 10 % Bis-Tris polyacrylamide gels (NuPage, Invitrogen) were equiloaded with 10–30μg of protein, electrophoresed at 200V, and electrotransferred to PVDF membranes. After blocking with 5% BSA, membranes were probed with primary antibodies to β-actin (8H10D10), p53 (7F5), PLC-γ (polyclonal), p-PLC-γ (polyclonal), Bcl-XL (54H6; all Cell Signaling), JNK (2C6), p-JNK (G9), Smad4 (polyclonal), p16 (polyclonal), c-Myc (9E10), CARD9 (polyclonal), Syk (polyclonal), p-Syk (polyclonal), Rb (C-15; all Cell Signalling), Dectin-1 (polyclonal; Abcam), Galectin-9 (polyclonal), and Dectin-1 Fc (fc-mdec1a; InvivoGen). Blots were developed by ECL (Thermo Scientific, Asheville, NC). RNA extraction was performed using the RNeasy Mini kit (Qiagen, Germantown, MD) as per manufacturer’s instructions. For Nanostring analysis, the nCounter mouse inflammation panel was employed using the nCounter Analysis System (both Nanostring, Seattle, Washington).
9
Western Blot Analysis of Cellular Signaling
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Cell or tissue homogenates (20 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, blots were probed with primary antibodies against p-LAT (ab4476), Ac-H3K18 (ab1191), PLCγ (ab76155) (all from Abcam), LAT (sc-53550, Santa Cruz Biotechnology), Sirt6 (#12486), p-Syk (#2717), Syk (#13198), p-PLCγ (#2821), p-PI3K (#4228), PI3K (#4257), p-Akt (#9271), Akt (#9272), p-ERK (#4370), ERK (#9102), p-p38 (#9211), p38 (#9212) (all from Cell Signaling Technology, Beverly, MA, USA), and HSP-90 (ADI-SPA-836-F, Enzo Life Sciences, Farmingdale, NY). After a brief washing, membranes were incubated for one hour with either horseradish peroxidase-conjugated goat anti-mouse IgG (ADI-SAB-100) or goat anti-rabbit IgG (ADI-SAB-300, Enzo Life Sciences) at room temperature. Antibody signals were detected using a Las-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).
10
Saponarin Modulates Inflammatory Responses
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Saponarin (purity > 98%) was obtained from Extrasynthese (Genay, France). Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), and trypsin-ethylenediaminetetraacetic acid (EDTA) were obtained from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Biowest (Kansas City, MO, USA), and lipopolysaccharide (LPS), monoclonal anti-DNP-IgE, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 4-nitrophenyl n-acetyl-b-d-glucosaminide (p-NAG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DNP-BSA was procured from Invitrogen (Carlsbad, CA, USA). Recombinant human TNF-α and IFN-γ were purchased from Peprotech (Rocky Hill, NJ, USA). Primary anti-bodies against ERK, JNK, p38, Lyn, Syk, PLCγ, STAT1, p-ERK, p-JNK, p-p38, p-STAT1, p-Lyn, p-Syk, p-PLCγ, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against TSLP was purchased from ABclonal (Woburn, MA, USA). Primary antibodies against FcεRIγ were purchased from LSBio (Seattle, WA, USA). The SensiFAST SYBR No-ROX kit mix was purchased from Biolines (Seoul, Korea). RAW264.7 (ATCC® TIB-71) and RBL-2H3 (ATCC® CRL-2256) cells were purchased from the American Type Culture Collection (ATCC). HaCaT cells were obtained from Byoung-Woo Lim of Konkuk University, Korea.
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