MA5-15174

antibody from Invitrogen Antibodies

Targeting: MAPK3 ERK1, p44erk1, p44mapk, PRKM3

Provider product page for MA5-15174

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [0]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [6]

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Product number: MA5-15174 - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-ERK1/ERK2 (Tyr204) Monoclonal Antibody (B.742.5)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: It is not recommended to aliquot this antibody. This antibody is not cross-reactive with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP kinase.
Reactivity: Human, Mouse, Rat, Drosophila, Porcine, Zebrafish
Host: Rabbit
Isotype: IgG
Antibody clone number: B.742.5
Vial size: 200 µL
Concentration: 98 µg/mL
Storage: -20°C

MAPK3 protein structure - MA5-15174 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: WB analysis was performed on whole cell extracts (30 µg) of A-431 (1), A-431 treated with EGF (200 ng/mL for 10 minutes) (2), A-431 treated with Afatinib followed by EGF (0.5uM of Afatinib for 6hrs, 200 ng/mL for 10 minutes) (3), MCF7 (4) and MCF7 treated with PMA (200 ng/mL for 10 minutes) (5), HeLa (6) and HeLa treated with PMA (200 ng/mL for 10 minutes) (7), NIH/3T3 (8) and NIH/3T3 treated with PMA (200 ng/mL for 10 minutes) (9). The blot was probed with Phospho-ERK1/ERK2 (Tyr204) Rabbit Monoclonal Antibody (Product # MA5-15174, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP (Product # A27036, 0.25 µg/mL, 1:4000 dilution). 44, 42 kDa band corresponding to Phospho-ERK1/ERK2 (Tyr204) was detected and increased upon EGF and PMA treatment across cell lines and pre-treatment with Afatinib (antagonist) resulted in inhibition of Phospho-ERK1/ERK2 (Tyr204) in A-431 cell line upon EGF treatment. Protein samples were electrophoresed using Novex NuPAGE 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock System (Product # EI0002) and Novex Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then wet transferred onto a nitrocellulose membrane. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: WB analysis was performed on whole cell extracts (30 µg) of A-431 (1), A-431 treated with EGF (200 ng/mL for 10 minutes) (2), A-431 treated with Afatinib followed by EGF (0.5uM of Afatinib for 6hrs, 200 ng/mL for 10 minutes) (3), MCF7 (4) and MCF7 treated with PMA (200 ng/mL for 10 minutes) (5), HeLa (6) and HeLa treated with PMA (200 ng/mL for 10 minutes) (7), NIH/3T3 (8) and NIH/3T3 treated with PMA (200 ng/mL for 10 minutes) (9). The blot was probed with Phospho-ERK1/ERK2 (Tyr204) Rabbit Monoclonal Antibody (Product # MA5-15174, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP (Product # A27036, 0.25 µg/mL, 1:4000 dilution). 44, 42 kDa band corresponding to Phospho-ERK1/ERK2 (Tyr204) was detected and increased upon EGF and PMA treatment across cell lines and pre-treatment with Afatinib (antagonist) resulted in inhibition of Phospho-ERK1/ERK2 (Tyr204) in A-431 cell line upon EGF treatment. Protein samples were electrophoresed using Novex NuPAGE 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock System (Product # EI0002) and Novex Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then wet transferred onto a nitrocellulose membrane. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of extracts from NIH/3T3 cells treated with UV light and PDGF, using Phospho-p44/42 MAPK pErk1/2 pThr202/Tyr204 monoclonal antibody (Product # MA5-15174) (upper), Phospho-p38 MAPK (Thr180/Tyr182) monoclonal antibody (middle), and Phospho-SAPK/JNK (Thr183/Tyr185) monoclonal antibody (lower).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4. Relative sensitivities of ERK antibodies toward the human and coral proteins. Immunoblot performed with anti-ERK and anti-phospho-ERK on total protein extracts of human fibroblasts (BJ) and Stylphora pistillata . The amount of protein loaded in each lane is indicated on the Supplementary Figure S4.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. Detection of ERK activity in corals. ( A ) Fluorescent immunoblot revealing activated (pERK) and total forms of ERK (ERK) present in Stylphora pistillata nubbins. Molecular weight standards in kilo Daltons (kDa) are indicated on the left side of the figure. ( B ) Immunoblot performed with ERK and pERK antibodies on protein extracts from coral nubbins incubated in the absence (Control) or presence of the MEK inhibitor U0126. Densitometric analysis of activated ERK intensities is presented on the right of the figure. The amido black total protein staining of the western blot membrane is shown as a loading control. The medians and standard deviations of three independent experiments are presented (***, p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. Induction of Spi-ERK phosphorylation by thermal and UV stresses. Immunoblot performed with ERK and pERK antibodies on protein extracts from coral nubbins incubated for 30 minutes in control (Cont.), thermal stress (T), UV stress (UV) or a combination of thermal and UV stresses (UV + T) conditions. The amido black total protein staining of the western blot membrane is shown as a loading control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Left: extracellular signal regulated kinase (ERK)-phosphorylation upon reperfusion after reperfusion of kidneys with (controlled hyperthermia (CH)) or without (control) exposure to controlled hyperthermia during machine perfusion. Single values ( n = 3), mean and SEM are shown; * P < 0.05. Right: Protein expression of HSP 72 after reperfusion of kidneys with (CH) or without (control) exposure to controlled hyperthermia during machine perfusion.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 MEK1/2-RK1/2 and STAT3 play indispensable, yet distinct roles in mammary epithelial cell migration. A , both the STAT3 inhibitor S3I-201 and the MEK1/2 inhibitor U0126 blocked migration of the MCF-10A cells induced by EGF. Shown are percentage of wound healing (in 16 h) in the presence or the absence of EGF and/or the inhibitor. n = 3, ** p < 0.001; Student's t test. B , both S3I-201 and U0126 abrogated EGF-induced upregulation of CTEN expression. Data shown are obtained from samples with 8 h of treatment. C , Western blot showing the dynamic changes in the TNS3 and CTEN protein during 8 h of EGF treatment. Total and phosphorylated ERK1/2 and STAT3 were detected using specific antibodies. n = 3, * p < 0.05, ** p < 0.001; Student's t test. D , distinct binding profiles for STAT3 and ERK1/2 to the cten gene promoter at different time points of EGF treatment, graphed from the corresponding ChIP-PCR data. E , U0126 abrogated ERK1/2 phosphorylation at all time points but inhibited STAT3-Tyr705 phosphorylation only at late time points of EGF stimulation ( i.e. , 3 and 8 h). F , S3I-201 blocked STAT3-Tyr705 phosphorylation but had no effect on ERK1/2 phosphorylation. ChIP, chromatin immunoprecipitation; CTEN, C-terminal tension; EGF, epithelial growth factor; ERK1/2, extracellular signal-regulated protein kinase 1/2; MEK1/2, mitogen-activated protein kinase kinase 1/2; STAT3, signal transducer and activator of transcription 3; TNS3, tensin-3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 The IL-6-STAT3-IL-6 feedback loop facilitates cell migration in response to continuous EGF treatment. A , IL-6 promoted EGF stimulated cell migration, whereas an anti-IL-6 antibody (alphaIL-6) significantly reduced cell migration. n = 3, * p < 0.05, Student's t test. B , IL-6 promoted STAT3-Y705 phosphorylation but had no effect on ERK1/2 phosphorylation. C , alphaIL-6 markedly reduced STAT3 phosphorylation but had no effect on ERK1/2 activation induced by EGF stimulation for 3 h. D and E , pharmacological inhibition of the EGFR, JAK, and Src kinases reduced or blocked STAT3 phosphorylation. F, relevant kinase inhibitors and anti-IL-6 antibody all decreased CTEN expression induced by EGF. G , nuclear translocation of STAT3 (STAT3-pY705) and ERK1/2 (pERK1/2) during continuous EGF stimulation. H , IL-6 transcription was regulated by ERK1/2 in the early phase ( e.g. , 10 min), but by STAT3 in the later phase ( e.g. , 3 h) of EGF stimulation. n = 3, * p < 0.05; ** p < 0.001, Student's t test. I , both S3I-201 and U0126 effectively blocked EGF-induced IL-6 expression in MCF-10A cells. EGF, epithelial growth factor; EGFR, epithelial growth factor receptor; ERK1/2, extracellular signal-regulated protein kinase 1/2; IL-6, interleukin-6; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3.