We stained single cell suspensions following standard procedures using fluorochrome-conjugated antibodies supplied by eBiosciences and directed against CD3 (145-2C11), TCRA/B (H57-597), CD4 (RM4-5), CD8a (53-6.7), PD1 (J43), ICOS (C398.4A), CD25 (7D4), CD69 (H1.2F3), CD86 (B7-2) (GL1), Ly-6G (Gr-1) (RB6-8C5), CD11b (M1/70), CD19 (1D3), ICOSL (HK5.3), BCL6 (K112-91) and FOXP3 (150D/E4). The antibody against CD45R (B220) (RA3-6B2) is from Miltenyi and the anti-IFNG (XMG1.2) was purchased from BD Biosciences. For detection of CXCR5, we used purified anti-CXCR5 antibody (2G8) from BD Biosciences and followed a three-step staining protocol as previously described (Johnston et al., 2009 (link)). Intracellular detection of BCL6 and FOXP3 were performed under standard conditions using the FOXP3 transcription factor staining buffer (eBiosciences) as directed by the manufacturer’s protocol. For flow cytometry detection of phosphorylated intracellular proteins, cells were fixed with 4% formaldehyde and permeabilized with 90% ice cold methanol and then incubated with phosphor-ERK (197G2), phosphor-AKT (D9EXP) and phosphor-S6 rabbit antibodies (D68F8) followed by secondary antibody staining using an anti-rabbit Alexa-647 antibody, all supplied by Cell Signaling. We acquired flow cytometry data using a FACS Canto cytometer (BD Biosciences) and analyzed them using FlowJo software (TreeStar).
Phosphor erk 197g2
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphor-Erk (197G2) is a monoclonal antibody that specifically recognizes the phosphorylated form of the Extracellular Signal-Regulated Kinase (Erk) protein. It can be used to detect and quantify the activation of the Erk signaling pathway in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitors cocktail, by Roche (2 mentions)
Enhanced chemiluminescence, by Merck Group (2 mentions)
Polyclonal rabbit anti human dnmt3a, by Sangon (2 mentions)
Jnk d 2, by Santa Cruz Biotechnology (2 mentions)
Phosphor jnk g9, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti mouse igg or anti rabbit igg antibodies, by Jackson ImmunoResearch (2 mentions)
Facscanto cytometer, by BD (1 mentions)
Fluorochrome conjugated antibodies, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (1 mentions)
Anti ifng xmg1.2, by BD (1 mentions)
Akt 11e7, by Cell Signaling Technology (1 mentions)
Erk 137f5, by Cell Signaling Technology (1 mentions)
Gapdh, by Sangon (1 mentions)
Phosphor akt, by Cell Signaling Technology (1 mentions)
3 protocols using phosphor erk 197g2
1
Multiparameter Flow Cytometry Immune Profiling
2
DNMT3A Knockout Effects on Cell Signaling
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DNMT3A KO and WT HEK293 cells of 1 ×10 6 were washed with PBS, lysed using 100 μL RAPA lysis buffer containing protease inhibitors cocktail (Roche; Penzberg, Germany), and separated by a 10% SDS-PAGE. After transferring onto a 0.45 μm PVDF membranes, immunoblotting was performed. For detection of DNMT3A deficiency, primary mouse monoclonal antibody against GAPDH (Sangon; Shanghai, China) and polyclonal rabbit-anti-human DNMT3A (Sangon) were used at 1:1000 dilution. For detection of MAPK and PI3K-Akt pathways, primary monoclonal antibodies against human Erk (137F5; Cell Signaling Technology; Danvers, MA, USA), phosphor-Erk (197G2; Cell Signaling Technology), JNK (D-2; Santa Cruz; Dallas, TX, USA), phosphor-JNK (G9; Cell Signaling Technology), Akt (11E7; Cell Signaling Technology), and phosphor-Akt (244F9; Cell Signaling Technology) were used. HRP-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (Jackson ImmunoResearch; PA, USA) were used for secondary antibodies. Signals were detected with enhanced chemiluminescence (Millipore; MA, USA) and visualized with a gel imaging system (Tanon; Shanghai, China).
3
DNMT3A Knockout Cell Proliferation
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DNMT3A KO and WT cells were cultured and seeded at 3×10 4 cells/well into 12-well plate. Cells were counted every 24 h for consecutive 6 days. And cell proliferation curves were compared between the two cell lines.
Western blot analysis DNMT3A KO and WT HEK293 cells of 1 ×10 6 were washed with PBS, lysed using 100 μL RAPA lysis buffer containing protease inhibitors cocktail (Roche; Penzberg, Germany), and separated by a 10% SDS-PAGE. After transferring onto a 0.45 μm PVDF membranes, immunoblotting was performed. For detection of DNMT3A de ciency, primary mouse monoclonal antibody against GAPDH (Sangon; Shanghai, China) and polyclonal rabbit-anti-human DNMT3A (Sangon) were used at 1:1000 dilution. For detection of MAPK and PI3K-Akt pathways, primary monoclonal antibodies against human Erk (137F5; Cell Signaling Technology; Danvers, MA, USA), phosphor-Erk (197G2; Cell Signaling Technology), JNK (D-2; Santa Cruz; Dallas, TX, USA), phosphor-JNK (G9; Cell Signaling Technology), Akt (11E7; Cell Signaling Technology), and phosphor-Akt (244F9; Cell Signaling Technology) were used. HRP-conjugated anti-mouse IgG or antirabbit IgG antibodies (Jackson ImmunoResearch; PA, USA) were used for secondary antibodies. Signals were detected with enhanced chemiluminescence (Millipore; MA, USA) and visualized with a gel imaging system (Tanon; Shanghai, China).
Western blot analysis DNMT3A KO and WT HEK293 cells of 1 ×10 6 were washed with PBS, lysed using 100 μL RAPA lysis buffer containing protease inhibitors cocktail (Roche; Penzberg, Germany), and separated by a 10% SDS-PAGE. After transferring onto a 0.45 μm PVDF membranes, immunoblotting was performed. For detection of DNMT3A de ciency, primary mouse monoclonal antibody against GAPDH (Sangon; Shanghai, China) and polyclonal rabbit-anti-human DNMT3A (Sangon) were used at 1:1000 dilution. For detection of MAPK and PI3K-Akt pathways, primary monoclonal antibodies against human Erk (137F5; Cell Signaling Technology; Danvers, MA, USA), phosphor-Erk (197G2; Cell Signaling Technology), JNK (D-2; Santa Cruz; Dallas, TX, USA), phosphor-JNK (G9; Cell Signaling Technology), Akt (11E7; Cell Signaling Technology), and phosphor-Akt (244F9; Cell Signaling Technology) were used. HRP-conjugated anti-mouse IgG or antirabbit IgG antibodies (Jackson ImmunoResearch; PA, USA) were used for secondary antibodies. Signals were detected with enhanced chemiluminescence (Millipore; MA, USA) and visualized with a gel imaging system (Tanon; Shanghai, China).
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