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.

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.

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-ra

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.

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.

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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 4ºC. 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