Nuclear and cytosolic proteins were extracted as previously described19 (link). Before protein extraction, RBL-2H3 cells (2 × 106/well in 6-well plates) were sensitized with anti-DNP IgE (50 ng/ml). After incubation overnight, the cells were pretreated with or without PD for 1 h and challenged with DNP-HSA (100 ng/ml). Then, cell extracts were prepared by detergent lysis procedure as described previously19 (link). Samples of protein (30 μg) were electrophoresed using 8–12% SDS-PAGE gel at 120 V for 90 min and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Immunodetection was carried out using a chemiluminescent substrate. The antibodies of IKK, phospho-IKK, NF-κB p65, phospho-NF-κB p65, PARP, IκBα, phospho-IκBα, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies of Syk, phospho-Syk, Lyn, phospho-Lyn, Gab2, phospho-Gab2, Akt, phospho-Akt, p38 MAPK, phospho-p38 MAPK, ERK, phospho-ERK, Nrf-2, and HO-1 were purchased from Cell Signaling Technology (Danvers, MA, USA).
Phospho gab2
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Gab2 is a laboratory product from Cell Signaling Technology. It is a specific antibody used to detect the phosphorylation of the Gab2 protein.
Automatically generated - may contain errors
Lab products found in correlation
Phospho syk, by Cell Signaling Technology (4 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
P38 mapk, by Cell Signaling Technology (2 mentions)
Phospho erk, by Cell Signaling Technology (2 mentions)
Phospho lyn, by Cell Signaling Technology (2 mentions)
Phospho p38 mapk, by Cell Signaling Technology (2 mentions)
Phospho plcγ2, by Cell Signaling Technology (2 mentions)
Phospho iκbα, by Santa Cruz Biotechnology (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Phospho nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Recombinant mouse m csf, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Bone resorption assay kit, by Cosmo Bio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
5 protocols using phospho gab2
1
Protein Extraction and Immunoblotting for RBL-2H3 Cells
2
Osteoclastogenesis Signaling Pathway
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Modified eagle’s medium (α-MEM), Dulbecco’s modified eagle’s medium (DMEM), trypsin-EDTA, and fetal bovine serum (FBS) were obtained from Gibco-BRL (Grand Island, NY). Recombinant mouse M-CSF and RANKL were obtained from Peprotech (Rocky Hill, NJ, USA). Rabbit polyclonal antibodies specific for phospho-ERK, phospho-JNK, phospho-p38, phospho-PLCγ2, phospho-Syk, IκB, phospho-IKK (Ser176/180), phospho-STAT3 (Ser727), and phospho-Gab2 were purchased from Cell Signaling Technology (Danvers, MS, USA). Mouse monoclonal antibodies specific for NFATc1, TRAF6, cathepsin K, c-Fos, STAT3, TBP, and NF-κB were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-actin antibody, β-glycerophosphate disodium salt hydrate, MSM, ascorbic acid phosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and L-glutamine were purchased from Sigma Chemical Co. (St. Louis, MO). A bone resorption assay kit was purchased from COSMO BIO Co. (Tokyo, Japan). The electrophoretic mobility shift assay (EMSA) kit and oligonucleotide probes (NF-κB) were purchased from Panomics (Redwood City, CA). The RNeasy mini kit was purchased from Qiagen (Hilden, Germany) and enhanced chemiluminescence (ECL) plus detection kit from Amersham Pharmacia Biotech. (Piscataway, NJ).
3
Phospho-Specific Antibody Sourcing for Cell Signaling
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Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), with the exception of anti-p44/42 MAP Kinase antibody and the following phospho-specific antibodies which were obtained from Cell Signaling Technologies (Danvers, MA, USA): Phospho Syk (Tyr525/526); Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204); Phospho-SAPK/JNK (Thr183/Tyr185); Phospho-p38 MAP Kinase (Thr180/Tyr182); Phospho-Src Family (Tyr416); Phospho-NFkB (Ser536); Phospho-Gab2 (Tyr452); Phospho-PLCγ2 (Tyr1217); Phospho-Akt (Ser473). The anti-human C12orf4 antibody and all reagents were obtained from Sigma-Aldrich (St Louis, MO, USA). Antiphosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology (Millipore, MA, USA). Alexa 488 conjugated anti-mouse IgG and Alexa 594 conjugated anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch laboratories (West Grove, PA, USA).
4
Analyzing Protein Interactions in MCF Cells
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Co-immunoprecipitation analyses of whole cells lysate or Cytoplasmic/nuclear protein solution were performed as previously described [54] (link) using Shp2 antibody [55] (link) to pull down the protein complex. Protein levels were determined by Western blot analysis. The antibodies to ERα (mc20, SC-542), IGF-1R (3B4, SC-462), Cyclin D1 (sc-753), Akt1 (B-1, SC-5298), p-Akt (Ser 473) (SC-798512), Erk (C-16, SC-16982), p-Erk (Thr 202/Tyr 204) (SC-16982), and β-Actin (C4, SC-47778) were purchased from Santa Cruz Company. The other antibodies were as follows: Gab2 (06-967, Millipore), Phospho-SHP-2 (Tyr580) (Cell Signal 3703), Phospho-Gab2 (Tyr452) (Cell signal 3882)
The GST pull-down assays were performed as previously described [54] (link). The plasmid pGEX4T1-Shp2 (a gift from Dr. Gensheng Feng at the University of California San Diego) was transformed into the engineering strain BL-21 and the GST-tagged Shp2 protein was expressed. Then, GST protein or GST-Shp2 fusion protein was incubated with MCF cell lysate at room temperature for 3 hours. After washing three times, the glutathione–agarose beads were resuspended in SDS sample buffer, and the protein interaction was analyzed with specific antibody.
The GST pull-down assays were performed as previously described [54] (link). The plasmid pGEX4T1-Shp2 (a gift from Dr. Gensheng Feng at the University of California San Diego) was transformed into the engineering strain BL-21 and the GST-tagged Shp2 protein was expressed. Then, GST protein or GST-Shp2 fusion protein was incubated with MCF cell lysate at room temperature for 3 hours. After washing three times, the glutathione–agarose beads were resuspended in SDS sample buffer, and the protein interaction was analyzed with specific antibody.
5
Immunoblot Analysis of Signaling Pathways
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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).
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