Total proteins were harvested from tissue and cell lines lysates using M-PER protein extraction reagent (Thermo Fisher, USA) respectively supplemented with proteinase and phosphatase inhibitors (Sigma, USA) according to standard protocol. Protein concentrations were determined using the BCA method followed by standard western immunoblotting of proteins using different primary antibodies: Anti-METTL3 (#195352, abcam, USA), Anti-METTL14 (#HPA038002, Sigma, USA), Anti-FTO (#ab124892, abcam, USA), Anti-P21 (#ab109199, abcam, USA), Anti-Phospho-AKT (#9271, Cell Signaling, USA), Anti-AKT (#9272, Cell Signaling, USA), Anti-Phospho ATM (#4526, Cell Signaling, USA), Anti-ATM (#2873, Cell Signaling, USA), Anti-Phospho-IR/IGF1R (#3021, Cell Signaling, USA), Anti-IGF1Rβ (#9750, Cell Signaling, USA), Anti-IRβ (#3025, Cell Signaling, USA), Anti-PDX1 (#5579, Cell Signaling, USA), Anti-Pan-Calcineurin A (#2614, Cell Signaling, USA), Anti-SHP-2 (#3397, Cell Signaling, USA), Anti-β-Actin (#4970, Cell Signaling, USA), Anti-αTubulin (#7291, abcam, USA). (Please see Reporting Summary for further details on antibodies used). The blots were developed using chemiluminescent substrate ECL (ThermoFisher, USA) and quantified using Image studio Lite Ver. 5.2 software (LICOR, USA).
Anti shp2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-SHP2 is a primary antibody that specifically binds to the SHP2 (PTPN11) protein. SHP2 is a non-receptor protein tyrosine phosphatase that plays a crucial role in various cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti jak2, by Cell Signaling Technology (3 mentions)
Anti shp 1, by Cell Signaling Technology (3 mentions)
Proteinase and phosphatase inhibitors, by Merck Group (2 mentions)
M per protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Anti mettl3, by Abcam (2 mentions)
Anti fto, by Abcam (2 mentions)
Anti p21, by Abcam (2 mentions)
Anti phospho atm, by Cell Signaling Technology (2 mentions)
Anti phospho ir igf1r, by Cell Signaling Technology (2 mentions)
Anti igf1rβ, by Cell Signaling Technology (2 mentions)
Anti irβ, by Cell Signaling Technology (2 mentions)
Anti pdx1, by Cell Signaling Technology (2 mentions)
Anti pan calcineurin a, by Cell Signaling Technology (2 mentions)
Image studio lite ver 5, by Thermo Fisher Scientific (2 mentions)
Anti mettl14, by Merck Group (2 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Anti α tubulin, by Abcam (2 mentions)
12 protocols using anti shp2
1
Quantitative Protein Analysis in Cell Lines
2
Western Blot Analysis of Signaling Proteins
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Cells were lyzed with RadioImmunoPrecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Villebon sur Yvette, France). Cell lysates were clarified by centrifugation at 14000 × g at 4°C for 15 min. For western blotting, proteins were separated by SDS-PAGE gels and transferred to a nitrocellulose membrane. Then, membranes were blocked with Tris buffered saline (TBS) (0.02 M Tris-HCl, 0.137 M NaCl, pH 7.4) containing 0.1% Tween (TBS-T) and 5% non-fat dry milk at room temperature during 1 hour and incubated overnight at 4°C with the following primary antibodies: anti-phospho-JAK2 (Santa Cruz Biotechnology), anti-GAPDH, anti-DDR1, anti-phospho-SHP2, anti-SHP2, anti-JAK2, anti-phospho-ERK1/2, anti-ERK1/2, anti-p21CIP1 (Cell signaling Technology, Saint Quentin Yvelines, France), anti-DDR2 (R&D systems, Lille, France), anti-RAGE (GeneTex, Irvine, CA). Membranes were washed with TBS-T and incubated with the corresponding peroxidase conjugated secondary antibody at room temperature for 1 hour. Chemiluminescent detection was realized by using an ECL Prime Kit (GE Healthcare, Orsay, France).
3
Western Blot Analysis of Protein Phosphorylation
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Total protein was extracted from cells or tissues using radioimmunoprecipitation assay lysis buffer containing 1 μmol/L/mL phenylmethanesulfonyl fluoride and phosphatase inhibitor. After measuring the protein concentration with the BCA Protein Assay Kit, an equal amount of protein extract (30 μg) was separated on an 8% or 10% SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked in TBS buffer with 0.1% Tween-20 (TBS-T) and 5% (w/v) nonfat milk for 2 h at room temperature. After blocking, the membrane was washed three times with TBS-T and then incubated with the primary antibodies at 4 °C overnight. Then the membrane was incubated with goat anti-mouse IgG secondary antibody or goat anti-rabbit IgG secondary antibody for 1 h at room temperature. Finally, the proteins were detected using an enhanced chemiluminescence system (Tanon, Shanghai, China) and analyzed with a digital imaging system (Tanon). Among the primary antibodies used, anti-p-SHP-2 (Tyr580, #3703) and anti-SHP2 (#3752) were purchased from Cell Signaling Technology (Danvers, MA, United States), anti-HA tag (#TA100012) was purchased from OriGene Technologies (Rockville, MD, United States), anti-β-Actin (#TA-09) was purchased from ZSBio (Beijing, China), and all secondary antibodies (#ZB-2301, #ZB-2305) were purchased from ZSBio.
4
Western Blot Analysis of Signaling Proteins
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Whole cell lysates were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were incubated with primary antibodies overnight at 4°C followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: anti-CREB, anti-Phospho-CREB (Ser133), anti-SHP-1, anti-Phospho-SHP1 (Tyr564), anti-SHP-2, anti-Phospho-SHP2 (Tyr580), anti-ITGB1, anti-ITGB3, p-CaMK2, CaMK2 (Cell Signaling Technology), anti-ANGPTL2 (R&D Systems), anti-CaMK1 (Abcam), anti-p-CaMK4 (Abmart), anti-CaMK4 (Genscript), anti-Flag (Sigma), anti-GAPDH, and anti-β-actin (Calbiochem). Anti-LILRB2-PE (eBioscience) and isotype control of IgG-PE were used to detect the expression of LILRB2 on the different lung cancer cell lines and analyzed by flow cytometry.
5
SHP2 Immunoprecipitation and Elution
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A cross-link immunoprecipitation kit was purchased from Thermo Pierce. Immunoprecipitation experiments were performed according to the manufacturer's instructions. Briefly, an SHP2 antibody (10 μg, Cell Signaling Technology, Beverly, US) was first incubated with protein A/G plus agarose resin (20 μl), and it was then covalently cross-linked onto the resin by incubation with 450 μM DSS at room temperature for 1 h. Effluent obtained after 24 h of cold storage was first precleared using control agarose resin to reduce nonspecific protein binding. Precleared effluent was incubated with protein A/G beads coupled to anti-SHP2 (Cell Signaling Technology, Beverly, US) at 4°C overnight. The effluent was eluted from the beads using antigen elution buffer for Western blot analysis.
6
Regulation of MAPK and AKT Signaling
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The indicated cells were incubated with DMSO, 5 µM vemurafenib, 10 µM SHP099, or the combination of 5 µM vemurafenib and 10 µM SHP099 for 0 h, 6 h, 24 h and 48 h. The cells were then lysed in RIPA lysis solution containing protease inhibitors for 30 min, and the protein concentration was determined using BCA reagent. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes and incubated overnight at 4 °C with primary antibodies. The next day, co-incubation with the secondary antibody of the corresponding species was carried out for 2 h. Chemiluminescence detection was performed using the ECL Protein Blotting Assay Kit (Human IgG) (Solarbio). The primary antibodies were used: anti-SHP2 (Cell Signaling Technology, CAT #3397), anti-p-SHP2 (Cell Signaling Technology, CAT #3751), anti-ERK (Cell Signaling Technology, CAT #4695), anti-p-ERK (Cell Signaling Technology, CAT #4370), anti-AKT (Cell Signaling Technology, CAT #4685), anti-p-AKT (Cell Signaling Technology, CAT #4060), anti-MEK (Cell Signaling Technology, CAT #4694), anti-p-MEK (Cell Signaling Technology, CAT #9127).
7
Western Blotting of Key Signaling Proteins
8
Quantitative Protein Analysis in Cell Lines
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Total proteins were harvested from tissue and cell lines lysates using M-PER protein extraction reagent (Thermo Fisher, USA) respectively supplemented with proteinase and phosphatase inhibitors (Sigma, USA) according to standard protocol. Protein concentrations were determined using the BCA method followed by standard western immunoblotting of proteins using different primary antibodies: Anti-METTL3 (#195352, abcam, USA), Anti-METTL14 (#HPA038002, Sigma, USA), Anti-FTO (#ab124892, abcam, USA), Anti-P21 (#ab109199, abcam, USA), Anti-Phospho-AKT (#9271, Cell Signaling, USA), Anti-AKT (#9272, Cell Signaling, USA), Anti-Phospho ATM (#4526, Cell Signaling, USA), Anti-ATM (#2873, Cell Signaling, USA), Anti-Phospho-IR/IGF1R (#3021, Cell Signaling, USA), Anti-IGF1Rβ (#9750, Cell Signaling, USA), Anti-IRβ (#3025, Cell Signaling, USA), Anti-PDX1 (#5579, Cell Signaling, USA), Anti-Pan-Calcineurin A (#2614, Cell Signaling, USA), Anti-SHP-2 (#3397, Cell Signaling, USA), Anti-β-Actin (#4970, Cell Signaling, USA), Anti-αTubulin (#7291, abcam, USA). (Please see Reporting Summary for further details on antibodies used). The blots were developed using chemiluminescent substrate ECL (ThermoFisher, USA) and quantified using Image studio Lite Ver. 5.2 software (LICOR, USA).
9
Signaling Pathway Regulation in Chondrocytes
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Recombinant mouse IL-1β was purchased from R&D Systems (Minneapolis, MN, United States). Lipofectamine 3000 reagent was obtained from Invitrogen (Waltham, MA, United States). Anti-SHP2, β-catenin, p38, p-p38, extracellular signal-regulated kinase (ERK), p-ERK, c-Jun N-terminal kinase (JNK), p-JNK, p65, p-p65, IκBα, p-IκBα, IκB kinase (IKK)-β and p-IKKα/β antibodies were supplied by Cell Signaling Technology (Beverly, MA, United States). MMP3, MMP13 and p-β-catenin(Y142) antibodies were obtained from Abcam (Cambridge, United Kingdom). p-β-catenin(S33/37/141), COL2A1, and AGGRECAN antibodies were purchased from Abclonal (Wuhan, China). GAPDH antibody was purchased from Boster (Wuhan, China) and secondary antibodies were obtained from the Jackson lab.
10
Western Blot Analysis of Tumor Cell Signaling
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Tumors were harvested and homogenized as described earlier59 (link). Protein lysates (20–30 μg) were loaded onto 10–12% SDS-PAGE gels. Electrophoresis, blocking, antibody incubation, and detection were performed as described59 (link). Blots were probed overnight at 4 °C with the respective antibodies. Anti-survivin (1:000), anti-Bcl-XL (1:1000), anti-p-c-Jun (1:1000), anti-Jun, anti-p-STAT3 (1:1000), anti-STAT3 (1:1000), anti-JAK2 (1:1000), anti-SHP2 (1:1000), anti-VEGF (1:500), anti-NRP1 (1:1000), anti-p-SHP1 (1:750), anti-SHP1 (1:750), and anti-β-actin (1:1000) HRP-conjugated antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-EphB4 (1:1000 or 0.5 μg/ml) antibody was purchased from Invitrogen (Carlsbad, CA, USA) or MilliporeSigma (Burlington, MA, USA), and anti-ephrin-B2 (1:750) was obtained from Abcam (Cambridge, MA, USA). Anti-EphA4 (1:750) antibody was obtained from Proteintech Group, Inc. (Rosemont, IL, USA). Anti-p-ephrinB2 (1:500) antibody was obtained from ThermoFisher Scientific. Goat anti-mouse, goat anti-rabbit, and donkey anti-goat horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Sigma (St. Louis, MO, USA) and used at a dilution of 1:3000.
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