- MA5-15228 - Invitrogen Antibodies MAPK9 antibody

No submitted references: Submit reference

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-JNK (green) in HeLa cells either left untreated (left panel) or treated with 25 µg/mL anisomycin (right panel) for 25 min. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a Phospho-SAPK/JNK pThr183/Tyr185 monoclonal antibody (Product # MA5-15228) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-JNK (green) in HeLa cells either left untreated (left panel) or treated with 25 µg/mL anisomycin (right panel) for 25 min. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a Phospho-SAPK/JNK pThr183/Tyr185 monoclonal antibody (Product # MA5-15228) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometric analysis of Phospho-JNK on Jurkat cells left untreated (green histogram) or treated with anisomycin (blue histogram). Cells were harvested, fixed with formaldehyde, permeabilized with methanol, and incubated with a Phospho-SAPK/JNK pThr183/Tyr185 monoclonal antibody (Product # MA5-15228) at a dilution of 1:200 for 1 hour at room temperature. As a negative control anisomycin-treated cells were stained with an isotype control (red histogram). Following staining with either MA5-15228 or isotype control, cells were stained with a fluorescently-conjugated anti-mouse IgG secondary antibody and analyzed on a flow cytometer.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometric analysis of Phospho-JNK on Jurkat cells left untreated (green histogram) or treated with anisomycin (blue histogram). Cells were harvested, fixed with formaldehyde, permeabilized with methanol, and incubated with a Phospho-SAPK/JNK pThr183/Tyr185 monoclonal antibody (Product # MA5-15228) at a dilution of 1:200 for 1 hour at room temperature. As a negative control anisomycin-treated cells were stained with an isotype control (red histogram). Following staining with either MA5-15228 or isotype control, cells were stained with a fluorescently-conjugated anti-mouse IgG secondary antibody and analyzed on a flow cytometer.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin immunoprecipitation analysis of Phospho-SAPK/JNK pThr183/Tyr185 was performed using cross-linked chromatin from 1 x 106 HCT116 colon carcinoma cells treated with serum for 0, 15, 30, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a Phospho-SAPK/JNK pThr183/Tyr185 monoclonal antibody (Product # MA5-15228). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions); the zigzag line represents an intron; and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin immunoprecipitation analysis of Phospho-SAPK/JNK pThr183/Tyr185 was performed using cross-linked chromatin from 1 x 106 HCT116 colon carcinoma cells treated with serum for 0, 15, 30, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a Phospho-SAPK/JNK pThr183/Tyr185 monoclonal antibody (Product # MA5-15228). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions); the zigzag line represents an intron; and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 KiPCs exhibited altered levels of cell cycle-regulated genes and mitogen-activated protein kinase (MAPK) kinase pathway members. ( A ) The cell cycle was analyzed by fluorescence-associated cell sorting after DNA staining with propidium iodide. M1: G0/G1, M2: S, and M3: G2/M phase. Percentages of cells in each cell cycle phase are shown in the bar graph. ( B ) Representative images of Bromodeoxyuridine (BrdU) staining (top) and percentages of BrdU-positive cells in the indicated groups (bottom). Magnification 100x. ( C ) qPCR analysis of cyclin-dependent kinase inhibitors. ( D ) qPCR analysis of cyclins and cyclin-dependent kinase-associated genes. ( E ) Cell cycle-related proteins and proliferation markers were measured by Western blot analysis. ( F ) Activation of three MAPKs was measured by Western blot analysis using phosphorylated protein-specific antibodies. Magnification 100x. All experiments were performed using at least three replicates, and the results are representative of three independent experiments. Data are presented as means +- standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way analysis of variance with Holm-Sidak multiple comparisons.

MA5-15228

Antibody data