61-9109-42

antibody from Invitrogen Antibodies

Targeting: MAPK3 ERK1, p44erk1, p44mapk, PRKM3

Provider product page for 61-9109-42

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
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Validations
Flow cytometry [1]
Other assay [7]

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Product number: 61-9109-42 - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-ERK1/2 (Thr202, Tyr204) Monoclonal Antibody (MILAN8R), PE-eFluor™ 610, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: This MILAN8R monoclonal antibody recognizes human and mouse extracellular signal-regulated kinases 1 and 2 (also known as ERK1/2, p44/p42, or MAPK3/1) when phosphorylated on T202/Y204. ERK1/2 belong to a family of conserved serine/threonine protein kinases known as mitogen-activated protein kinases (MAPKs) that are involved in many cellular programs such as proliferation, differentiation, motility, and survival. ERK1/2 signaling is activated in response to numerous extracellular stimuli including mitogens, growth factors, and cytokines. The primary activators of ERK1/2 are MEK1 and MEK2 which act by phosphorylating the activation loop residues T202/Y204 and T185/Y187 in ERK1 and ERK2, respectively. Several downstream targets of ERK1/2 have been identified, including p90RSK and the transcription factor Elk-1. ERK1/2 are negatively regulated by MAPK phosphatases, known as DUSPs or MKPs, as well as by chemical inhibitors of MEK including U0126 and PD98059. Disruption of the ERK pathway is common in many types of cancer. Specificity of this MILAN8R clone was determined by ELISA, flow cytometry, and western blotting. Applications Reported: This MILAN8R antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This MILAN8R antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (0.5 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Staining Protocol: We recommend using Protocol C: Two-step protocol: Fixation/Methanol. Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins and Protocol B: One-step protocol: intracellular (nuclear) proteins cannot be used. All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the Best Protocols Section under the Resources tab online. PE-eFluor® 610 can be excited with laser lines from 488-561 nm and emits at 607 nm. We recommend using a 610/20 band pass filter (equivalent to PE-Texas Red®). Please make sure that your instrument is capable of detecting this fluorochome. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 607 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: MILAN8R
Vial size: 100 Tests
Concentration: 5 µL/Test
Storage: 4° C, store in dark, DO NOT FREEZE!

MAPK3 protein structure - 61-9109-42 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Correlation between MHC II induction and basal phosphorylated ERK1/2 levels in murine NSCLC cells. Mouse NSCLC lines were cultured for 48 hours, and analyzed for basal levels of intracellular pERK1/2 by flow cytometric analysis. (A) Relative pERK1/2 levels in four murine NSCLC cell lines, including (B) representative histograms. Data are from two independent experiments, with n=3 replicates total. Statistical comparisons of pERK levels were focused on comparison of oncogene-matched cancer cell lines; pERK levels were not significantly different between CMT167 and EA1 cells. MFI is x10 6 . Graphs show mean+-SEM. Flow cytometry data analyzed singlets with MFI data visualized x10 6 values. LLC, Lewis lung carcinoma; MFI, median fluorescent intensity; NSCLC, non-small cell lung cancer.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 4.1R deficiency regulated the function of CAR T cells via ERK signaling pathway. a Mock T, NKG2D-CAR T, NC-NKG2D-CAR T, and KD2-NKG2D-CAR T were co-incubated with PANC28 at a 9:1 ratio for 16 h. The expression of p-ERK was detected by flow cytometry (left), and the percentage of p-ERK-positive T cells was statistically analyzed (right) ( n = 3). b Line plots displayed the cytotoxicity of NC-NKG2D-CAR T and KD2-NKG2D-CAR T against PANC28 at a different effector to target ( E : T ) ratios for 16 h in the absence and presence of 10 muM U0126. c NC-NKG2D-CAR T and KD2-NKG2D-CAR T were co-incubated with PANC28 at a different effector to target ( E : T ) ratios for 7 days in the absence and presence of 10 muM U0126. CFSE dilution was used as a measure of cell proliferation (left), and MFI was calculated (right) ( n = 3). NC-NKG2D-CAR T and KD2-NKG2D-CAR T were co-incubated with PANC28 at a different effector to target ( E : T ) ratios for 16 h in the absence and presence of 10 muM U0126. The expression of CD69 ( d ), Gzm B ( e ), and TIM-3 ( f ) was detected by flow cytometry (left). MFI and percentage were statistically analyzed and shown in column chart (middle and right) ( n = 3). Data were representative of three independent experiments. ** P < 0.01, *** P < 0.001, NS not significant.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 4 Elevated NFkappaB p65 but not ERK1/2 activity on miR-146a and miR-155 knockout. A) Representative FACS scatter plots showing a right-shift of 30 min LPS-stimulated (1 mug / ml) compared to mock-treated monocytes stained with phospho-p65 antibody (PE-channel). B) Representative histogram plots showing an increased right-shift of miR-146a and miR-155 deficient compared to control or MALAT1 deficient monocytes after 30 min LPS-stimulation (1 mug / ml) and staining with a phospho-p65 antibody (PE-channel). C) Fold change in phospho-p65 signal in monocytes stimulated with LPS (1 mug / ml) for 15, 30 or 100 min compared to mock-treatment (ctrl) in wild-type (WT) or the indicated ncRNA knockout (KO) cells. All fold-changes are relative to the respective WT mock control. D-F) Same as A-C) but with phospho-ERK1/2 staining (APC-channel). Statistical significance was determined by a one-way ANOVA test with multiple comparisons (* p