Anti-MAPK6/ERK3 antibody [EP1720Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MAPK6/ERK3 antibody [EP1720Y] (AB53277)

Unpurified ab53277 (1 : 50) staining human MAPK6/ERK3 in human breast carcinoma tissue by immunohistochemistry using paraffin embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MAPK6/ERK3 antibody [EP1720Y] (AB53277)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney carcinoma tissue sections labeling MAPK6/ERK3 with Purified ab53277 at 1 : 100 dilution (13.52 μg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Purified)ImmunoHistoProbe one step HRP Polymer (ready to use) was used for detection. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Lab

Western blot - Anti-MAPK6/ERK3 antibody [EP1720Y] (AB53277)

Lanes 1-3 : Merged signal (red and green). Green - ab53277 observed at 90 kDa. Red - loading control ab8245 observed at 36 kDa.

ab53277 Anti-MAPK6/ERK3 antibody [EP1720Y] was shown to specifically react with MAPK6/ERK3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264910 (knockout cell lysate ab257526) was used. Wild-type and MAPK6/ERK3 knockout samples were subjected to SDS-PAGE. ab53277 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MAPK6/ERK3 antibody [EP1720Y] (ab53277) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MAPK6 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MAPK6 (ERK3) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mapk6-erk3-knockout-hela-cell-line-ab264910'>ab264910</a>)

Lane 3:

2.4G2 (Rat B cell lymphoma B lymphocyte) cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 83 kDa

Observed band size: 90 kDa

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• WB

Lab

Western blot - Anti-MAPK6/ERK3 antibody [EP1720Y] (AB53277)

Lanes 1 - 4 : Merged signal (red and green). Green - ab53277 observed at 90 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab53277 was shown to react with MAPK6 in HAP1 wild-type cells in Western blot. Loss of signal was observed when MAPK6 knockout sample was used. HAP1 wild-type and MAPK6 knockout whole cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab53277 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MAPK6/ERK3 antibody [EP1720Y] (ab53277) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

MAPK6 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

A431 whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 83 kDa

Observed band size: 90 kDa

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• WB

Lab

Western blot - Anti-MAPK6/ERK3 antibody [EP1720Y] (AB53277)

The molecular weight observed is consistent with what has been described in PMID : 30642948 and 30166347

All lanes:

Western blot - Anti-MAPK6/ERK3 antibody [EP1720Y] (ab53277) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

Rat brain lysates at 15 µg

Lane 3:

C2C12 (Mouse myoblasts myoblast) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 83 kDa

Observed band size: 105 kDa

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• WB

CiteAb

Western blot - Anti-MAPK6/ERK3 antibody [EP1720Y] (AB53277)

MAPK6/ERK3 western blot using anti-MAPK6/ERK3 antibody [EP1720Y] ab53277. Publication image and figure legend from He, J., Huang, Z., et al., 2020, Mol Cancer, PubMed 31992303.

ab53277 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab53277 please see the product overview.

Inhibition of phosphorylation of p38/MAPK reverses cell survival induced by circ-MAPK4. (a) U373 cells transfected with siRNA negative control, circ-MAPK4 siRNA-1 and siRNA-2 were treated with or without p-p38/MAPK inhibitor (SB203580). Inhibition efficiency of p-p38/MAPK inhibitor was accessed by testing phosphorylation and total protein levels of p38/MAPK and ERK using western blot assays. (b, c) CCK-8 and colony formation assays were performed to reveal cell survival of these U373 cells. (d) Apoptosis assays were performed to reveal apoptosis levels of these U373 cells. (e) The western blot assays were used to evaluate effect of p-p38/MAPK inhibitor on protein expression levels of cleaved form of caspase-9, caspase-7, caspase-3, PARP1 in these U373 cells. The data were summarized as bar graph. Data are the means ± SEM of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001

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