Anti-ERK1 + ERK2 antibody

• ICC

Unknown

Immunocytochemistry - Anti-ERK1 + ERK2 antibody (AB17942)

Immunofluorescent analysis of ERK1 + ERK2 Antibody was done on 70% confluent log phase U87-MG cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ab17942 at 1 : 250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody at a dilution of 1 : 400 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Alexa Fluor 594 Phalloidin. Panel d is a merged image showing cytoplasmic and nuclear localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 + ERK2 antibody (AB17942)

Immunohistochemistry analysis of ERK1/2 (pan) showing staining in the cytoplasm and nucleus of paraffin-embedded human colon carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab17942 diluted in 3% BSA-PBS at a dilution of 1 : 20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 + ERK2 antibody (AB17942)

Immunohistochemistry analysis of ERK1/2 (pan) showing staining in the cytoplasm and nucleus of paraffin-embedded human breast carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab17942 diluted in 3% BSA-PBS at a dilution of 1 : 100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERK1 + ERK2 antibody (AB17942)

Immunohistochemistry analysis of ERK1/2 (pan) showing staining in the cytoplasm and nucleus of paraffin-embedded mouse stomach tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab17942 diluted in 3% BSA-PBS at a dilution of 1 : 20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

• WB

AbReview11057****

Western blot - Anti-ERK1 + ERK2 antibody (AB17942)

The tissue was harvested seven days post surgery, sonicated with RIPA buffer and the protein estimate made by Lowry. A 10% SDS-PAGE gel was run for 1.5 hr at 100V and transfered to PVDF membrane for 1.5 hr at 274 mA. The blot was blocked with 5% BSA for 1 hour at 23°C. The primary antibody was incubated with the blot for 18 hours at 4°C.

All lanes:

Western blot - Anti-ERK1 + ERK2 antibody (ab17942) at 1/1000 dilution

Lane 1:

Rat spinal cord tissue homogenate from animals that underwent Sham surgery at 20 µg

Lanes 2 - 3:

Rat spinal cord tissue homogenate from animals that underwent L5 nerve transection at 20 µg

Secondary

All lanes:

HRP conjugated goat anti-rabbit antibody at 1/3000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa,44 kDa

true

Exposure time: 5min

This image is courtesy of an anonymous Abreview

• WB

Unknown

Western blot - Anti-ERK1 + ERK2 antibody (AB17942)

Western Blot for ab17942.

Extracts prepared from PC12 cells not stimulated (-), or stimulated with NGF (+) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to nitrocellulose. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4C, then were incubated with ERK1&2 pan antibody for two hours at room temperature in a 3% BSA-TBST buffer. After washing, membranes were incubated with goat anti-rabbit IgG alkaline phosphatase.

These data show that ab17942 ERK1&2 antibody allows the total amount of ERK1&2 to be measured.

All lanes:

Western blot - Anti-ERK1 + ERK2 antibody (ab17942)

Predicted band size: 41 kDa

false

• WB

PubMed

Western blot - Anti-ERK1 + ERK2 antibody (AB17942)

Western blot analysis of Mice retinas (40-50μg/lane) labelling with anti-ERK1/2 at 1∶300 (ab17942) and mouse monoclonal anti-phosphorylated ERK1/2 at 1∶300 (ab50011), in 5% nonfat milk in TBST overnight at 4°C. HRP conjugated antibodies were used as the secondary antibodies.

Data is expressed as percentage change in phosphorylated ERK1/2 (p-ERK1/2) over total ERK1/2 (t-ERK1/2) calculated in control and diabetic mice maintained with and without Edaravone treatment

Results are expressed as mean±SD. Values obtained from Normal group are considered as 100%. *P<0.05, ***P<0.001 vs. Normal, ^#P<0.05 vs. Edaravone

All lanes:

Western blot - Anti-ERK1 + ERK2 antibody (ab17942)

Predicted band size: 41 kDa

true

Image from PLoS One. 2014; 9(6): e99219. Fig3A, doi: 10.1371/journal.pone.0099219 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

CiteAb

Western blot - Anti-ERK1 + ERK2 antibody (AB17942)

Western Blotting using Anti-ERK1 + ERK2 antibody, ab17942. Publication image from Birtley, J. R. et al., 2019, Nat Commun, 31316070. Legend direct from paper.

R65L, N70H, and P95R mutants interact differently with AXL and FGFR1. SKOV3 expressing an empty vector (CTRL), wild-type (OPCML) or mutant (R65L, N70H and P95R) OPCML were simulated for 30 min or 3 h with GAS6 (a) or FGF1 (b), and the interaction between OPCML and AXL (a) or OPCML and FGFR1 (b) was analyzed and quantified by Proximity Ligation Assay (DuoLink). The values from three independent experiments with five images each are shown all together. The bars indicate the mean ± s.e.m. Student t-test : *p < 0.05, ***p < 0.001. c, d Mammalian 2-hybrid assay. Cells were transfected with the indicated single constructs and the relative empty plasmids (not indicated), or with both plasmids expressing OPCML or P95R plus AXL (c) or plus FGFR1 (d). The bars indicate the mean ± s.e.m. calculated from the values of all the repeats from three independent experiments. Student t-test compared to wild-type OPCML plus AXL (c) or OPCML plus FGFR1 (d) : ***p < 0.001, ****p < 0.0001. e Gap closure assay. SKOV3 cells expressing the indicated wild-type and mutant OPCML were serum starved and then left without stimulation (−) or stimulated with GAS6 or FGF1 as indicated. Migration was imaged after 15 h, and the migration front is highlighted by the dotted lines. Scale bar = 200 µm. f, g SKOV3 cells expressing the indicated wild-type and mutant OPCML were serum-starved and then stimulated with GAS6 (f) or FGF1 (g) for 30 min or 3 h. Cell lysates were analyzed by western blotting for the expression of total and phosphorylated AKT and ERK. Calnexin was used as loading control. h, i SKOV3 cells expressing the indicated wild-type and mutant OPCML were treated with the AXL inhibitor R428 for 2 days to assess the IC50 (h) or for 1 day to measure apoptosis (i). Student t-test compares CTRL and mutants to wild-type OPCML : ***p < 0.001, ****p < 0.0001. Both graphs show the mean ± s.e.m. of three independent experiments

false

• WB

CiteAb

Western blot - Anti-ERK1 + ERK2 antibody (AB17942)

Western Blotting using Anti-ERK1 + ERK2 antibody, ab17942. Publication image from Birtley, J. R. et al., 2019, Nat Commun, 31316070. Legend direct from paper.

R65L, N70H, and P95R mutants interact differently with AXL and FGFR1. SKOV3 expressing an empty vector (CTRL), wild-type (OPCML) or mutant (R65L, N70H and P95R) OPCML were simulated for 30 min or 3 h with GAS6 (a) or FGF1 (b), and the interaction between OPCML and AXL (a) or OPCML and FGFR1 (b) was analyzed and quantified by Proximity Ligation Assay (DuoLink). The values from three independent experiments with five images each are shown all together. The bars indicate the mean ± s.e.m. Student t-test : *p < 0.05, ***p < 0.001. c, d Mammalian 2-hybrid assay. Cells were transfected with the indicated single constructs and the relative empty plasmids (not indicated), or with both plasmids expressing OPCML or P95R plus AXL (c) or plus FGFR1 (d). The bars indicate the mean ± s.e.m. calculated from the values of all the repeats from three independent experiments. Student t-test compared to wild-type OPCML plus AXL (c) or OPCML plus FGFR1 (d) : ***p < 0.001, ****p < 0.0001. e Gap closure assay. SKOV3 cells expressing the indicated wild-type and mutant OPCML were serum starved and then left without stimulation (−) or stimulated with GAS6 or FGF1 as indicated. Migration was imaged after 15 h, and the migration front is highlighted by the dotted lines. Scale bar = 200 µm. f, g SKOV3 cells expressing the indicated wild-type and mutant OPCML were serum-starved and then stimulated with GAS6 (f) or FGF1 (g) for 30 min or 3 h. Cell lysates were analyzed by western blotting for the expression of total and phosphorylated AKT and ERK. Calnexin was used as loading control. h, i SKOV3 cells expressing the indicated wild-type and mutant OPCML were treated with the AXL inhibitor R428 for 2 days to assess the IC50 (h) or for 1 day to measure apoptosis (i). Student t-test compares CTRL and mutants to wild-type OPCML : ***p < 0.001, ****p < 0.0001. Both graphs show the mean ± s.e.m. of three independent experiments

false

• WB

CiteAb

Western blot - Anti-ERK1 + ERK2 antibody (AB17942)

Western Blotting using Anti-ERK1 + ERK2 antibody, ab17942. Publication image from Birtley, J. R. et al., 2019, Nat Commun, 31316070. Legend direct from paper.

R65L, N70H, and P95R mutants interact differently with AXL and FGFR1. SKOV3 expressing an empty vector (CTRL), wild-type (OPCML) or mutant (R65L, N70H and P95R) OPCML were simulated for 30 min or 3 h with GAS6 (a) or FGF1 (b), and the interaction between OPCML and AXL (a) or OPCML and FGFR1 (b) was analyzed and quantified by Proximity Ligation Assay (DuoLink). The values from three independent experiments with five images each are shown all together. The bars indicate the mean ± s.e.m. Student t-test : *p < 0.05, ***p < 0.001. c, d Mammalian 2-hybrid assay. Cells were transfected with the indicated single constructs and the relative empty plasmids (not indicated), or with both plasmids expressing OPCML or P95R plus AXL (c) or plus FGFR1 (d). The bars indicate the mean ± s.e.m. calculated from the values of all the repeats from three independent experiments. Student t-test compared to wild-type OPCML plus AXL (c) or OPCML plus FGFR1 (d) : ***p < 0.001, ****p < 0.0001. e Gap closure assay. SKOV3 cells expressing the indicated wild-type and mutant OPCML were serum starved and then left without stimulation (−) or stimulated with GAS6 or FGF1 as indicated. Migration was imaged after 15 h, and the migration front is highlighted by the dotted lines. Scale bar = 200 µm. f, g SKOV3 cells expressing the indicated wild-type and mutant OPCML were serum-starved and then stimulated with GAS6 (f) or FGF1 (g) for 30 min or 3 h. Cell lysates were analyzed by western blotting for the expression of total and phosphorylated AKT and ERK. Calnexin was used as loading control. h, i SKOV3 cells expressing the indicated wild-type and mutant OPCML were treated with the AXL inhibitor R428 for 2 days to assess the IC50 (h) or for 1 day to measure apoptosis (i). Student t-test compares CTRL and mutants to wild-type OPCML : ***p < 0.001, ****p < 0.0001. Both graphs show the mean ± s.e.m. of three independent experiments

false