IHC were performed to examine cell proliferation marker as Ki67, pHH3, PCNA, and pERK in tumor tissues. IHC were also performed to examine inflammatory cell marker CD56. After being processed for paraffin embedding, 5 μm sections of tissue samples were prepared. Sections were boiled in 10 mM sodium citrate buffer (PH 6.0) for 20 min, and incubated in 0.3% hydrogen peroxide for 20 min and then blocked with 5% BSA for 1 h. Then incubated anti-Ki67 (Cell Signaling Technology, IHC, 1:400), anti-Phospho-Histone H3(Cell Signaling Technology, IHC, 1:50), anti-PCNA (Cell Signaling Technology, IHC, 1:4000) and anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, IHC,1:400), anti-CD56(Cell Signaling Technology, IHC, 1:800) antibodies overnight at 4 °C, followed by biotinylated secondary antibodies and DAB detection.
Anti phospho p44 42 mapk erk1 2 thr202 tyr204
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) is a primary antibody that specifically recognizes the dually phosphorylated forms of p44/42 MAPK (Erk1/2) at Thr202 and Tyr204. It is used to detect the activation of the MAPK/ERK signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti p44 42 mapk erk1 2, by Cell Signaling Technology (19 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (5 mentions)
Anti phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (5 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti p38 mapk, by Cell Signaling Technology (4 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Anti β actin ac 15, by Merck Group (2 mentions)
Anti pan akt, by Cell Signaling Technology (2 mentions)
Anti pd l1, by Abcam (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Ab11994, by Abcam (2 mentions)
Ab45039, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti phospho akt thr308, by Cell Signaling Technology (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Anti β actin c4, by Santa Cruz Biotechnology (2 mentions)
Anti granzyme k, by Thermo Fisher Scientific (2 mentions)
Anti phospho stat3 tyr705 d3a7, by Cell Signaling Technology (2 mentions)
49 protocols using anti phospho p44 42 mapk erk1 2 thr202 tyr204
1
Immunohistochemical Analysis of Tumor Cell Markers
2
Western Blotting Analysis of Protein Targets
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Western blotting (WB) was performed according to previous reports [13 (link)]. The primary antibodies used were anti-β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), anti-NRAS (#10724-1-AP, Proteintech, Chicago, IL, USA), anti-DOHH (#254866, Abcam, Cambridge, MA, USA), anti-HIST1H2AC (#15953-1-AP, proteintech), anti-TAF6 (#ab76922, Abcam), anti-p44/42 MAPK (Erk1/2) (#4695, Cell Signaling Technology), and anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9101, Cell Signaling Technology). Each primary antibody was diluted to 1:1000 and incubated overnight. The secondary antibody, diluted to 1:5000, was incubated at room temperature for 1 h. Protein signals were detected using ChemiDoc XRS Plus Systems (Bio-Rad, Hercules, CA, USA) after a 5 min reaction with the ECL substrate (Bio-Rad). Original images of Western blot analysis please see Supplementary File S1 .
3
Monoclonal Antibody-Based Cell Analysis
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MRG1 is a home-made mouse monoclonal antibody specific to human CEACAM1 [24] (link). Other antibodies used were anti–phospho-p44/42 MAPK (ERK1/2) Thr 202/Tyr 204 (Cell Signaling), anti–p44/42 MAPK (ERK1/2) (Cell Signaling), and anti-ETS1 antibody [1G11] 10936 (Abcam). FITC-conjugated goat anti-mouse polyclonal antibodies were used as secondary reagent in FACS assays (Jaxon Immunoresearch, USA).
4
Co-immunoprecipitation Assay for Protein-Protein Interactions
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Cells were lysed in 20 mM Hepes buffer, 10 mM KCl, 1 mM EDTA, 0.2% NP-40, 10% Glycerol44 (link), 59 (link) and co-immunoprecipitation studies were performed as previously described.44 (link) Details can be found in Supplementary Information . The following antibodies were used: mouse monoclonal anti-β-Actin (AC-15, Sigma-Aldrich, St Louis, MO, USA), mouse anti-HSP90 (Heat Shock Protein 90), rabbit polyclonal anti-KLF4 (sc-20691), rabbit polyclonal anti-p21 (C-19; sc-397), mouse monoclonal anti-p53 (DO-1; sc-126), mouse anti-Myc (9E10), mouse monoclonal anti-Caspase-3 (E-8; sc-7272), goat anti-Fibrillarin (D-14; sc-11336), goat anti-GAPDH (V-18; sc-20357), and mouse anti-BRAF wt (sc-5284) (Santa Cruz Biotechnology), rabbit polyclonal anti-BAX, rabbit polyclonal anti-BCL2, rabbit polyclonal anti-E2F1, rabbit polyclonal anti-poly ADP-ribose polymerase, rabbit polyclonal anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), rabbit polyclonal anti-Cleaved Caspase 3 (Asp175) (Cell Signaling Technologies, Danvers, MA, USA). Chemiluminescent detection was used.
5
Cellular Signaling Pathway Analysis
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The following antibodies were used: anti-Panras C-4 (sc-166691, Santa Cruz Biotechnology), anti-RASGTP (26909, Neweast Bioscienes), anti-SNAP23 (10825-1-AP, Proteintech), anti-SNAP29 (12704-1-AP, Proteintech), anti-VAMP3 (10702-1-AP, Proteintech), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4377, Cell Signaling Technologies), anti-p44/42 Erk1/2 (9107, Cell Signaling Technologies), anti-Phospho-AKT (Ser473) (4060, Cell Signaling Technologies), anti-AKT(pan) (2920, Cell Signaling Technologies).
6
Protein Analysis via Western Blotting
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Protein extraction, denaturation, and Western blotting were performed as previously described (29 (link), 60 (link), 61 (link)). Membranes were blotted with anti–phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, Cell Signaling) and anti–p44/42 MAPK (Erk1/2) (4695, Cell Signaling). Images were captured with ImageQuantLAS 4000 (GE Healthcare Life Sciences). For detection of D2AP11-TCE expression, goat anti-human IgG F(ab′)2 (109-005-006, Jackson ImmunoResearch Laboratories Inc.) and donkey anti-goat secondary antibodies (926-32214, LI-COR) were used, and membranes were scanned using a LI-COR Odyssey CLx imager.
7
Isoflavone and E2 Signaling Pathways
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Cultured cells were exposed to isoflavones or E2 for 30 min then rinsed three times with PBS, fixed with 4% PFA, and blocked with 2% FBS. Cells were then incubated with rabbit monoclonal anti-phospho-Akt (Ser473) (D9E) XP (1:200; Cell Signaling, MA, USA), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:200; Cell Signaling), or anti-phospho-Rac1/Cdc42 (Ser71) (1:200; Cell Signaling) antibodies, followed by CytoPainter Phalloidin-iFluor 594 reagent (Abcam) and donkey anti-rabbit IgG (H+L) secondary antibodies, Alexa Fluor® 488 conjugate (1:200; Thermo Fisher Scientific, Inc, Waltham, MA, USA). Cell nuclei were also stained with DAPI. The cells were then inspected using a laser confocal scanning microscope (Zeiss LSM 880, Carl Zeiss Microscopy GmbH).
8
Protein Extraction and Western Blot Analysis
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For whole protein extracts, tumor tissues or cell pellets were homogenized in RIPA buffer (Sigma) containing protease inhibitor cocktail (Roche), incubated on ice for 30 minutes, and then centrifuged for 15 minutes at 4°C at 12,000g. Primary antibodies used for Western blot include anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, 1:2000), anti-p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 1:2000), anti-Phospho-MEK (Cell Signaling Technology, 1:2000), anti-MEK (Cell Signaling Technology, 1:2000), anti-actin (Cell Signaling Technology, 1:2000), anti-PD-L1 (Abcam, Cambridge, MA, 1:100).
9
Immunoblotting for Protein Detection
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Proteins were separated through electrophoresis in 12% SDS-PAGE gel and electro-transferred to Hybond-P PVDF membranes (Amersham Biosciences) for blotting. Immunodetection was carried out using standard methods. The anti-HA, anti-FLAG (Santa Cruz Biotechnology) and anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibodies (Cell Signaling Technology) were used at a dilution of 1:1,000. HRP-conjugated secondary antibody (Sigma-Aldrich) was used at a dilution of 1:20,000. Blots were visualized with SuperSignal West Pico Chemiluminescent Substrates (Thermo Scientific) following the manufacturer’s instructions.
10
Hippocampal DUSP6 Protein Analysis
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The CA1 region of hippocampus was dissected from naïve DUSP6+/+ and DUSP6−/− mice, as well as from DUSP6+/+ and DUSP6−/− mice 24 h after tGCI. Tissues were placed in a Teflon-glass homogenizer and homogenized in ice-cold RIPA buffer containing 5 mM EDTA, 1 mM PMSF, 10 nM Pepstatin A, and 1X Halt Protease and Phosphatase inhibitors. The homogenate was centrifuged at 20,000 X g for 10 min at 4 °C. The supernatant was collected for Western blotting using rabbit anti-phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) (1:1000; Cell Signaling, Danvers, MA, USA, Cat# 4370) and anti-p44/42 MAPK (Erk1/2) (1:1000; Cell Signaling, Cat# 9102).
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