- MA5-15016 - Invitrogen Antibodies MAP2K2 antibody

Submitted references Yersinia pseudotuberculosis YopJ Limits Macrophage Response by Downregulating COX-2-Mediated Biosynthesis of PGE2 in a MAPK/ERK-Dependent Manner.

Sheppe AEF, Santelices J, Czyz DM, Edelmann MJ
Microbiology spectrum 2021 Sep 3;9(1):e0049621

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed by loading 30 µg of A-431 (Lane 1), A-431 treated with EGF (200 ng/mL for 10 minutes) (Lane 2), A-431 EGFR KO (Lane 3) A-431 EGFR KO treated with EGF (200 ng/mL for 10 minutes) (Lane 4) whole cell extracts using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex Protein Standard (Product # LC5800). Proteins were transferred to a nitrocellulose membrane using iBlot® Dry Blotting System (IB21001) and blocked with 5% skim milk for 1 hour at room temperature. The blot was probed with Anti-Phospho-MEK1/MEK2 (Ser217, Ser221) Rabbit Monoclonal Antibody (Product # MA5-15016, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 45 kDa band corresponding to Phospho-MEK1/MEK2 (Ser217, Ser221) was detected and observed to be increased upon EGF treatment in A-431 control cell line (lane1 and 2) and not in EGFR knockout cell line (lane 3 and 4).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: WB analysis was performed on whole cell extracts (30 µg lysate) of A549 (1), A549 treated with EGF (200 ng/mL for 10 minutes) (2), A-431 (3), A-431 treated with EGF (200 ng/mL for 10 minutes) (4) and A-431 treated with Afatinib followed by EGF (0.5 uM of Afatinib for 6 hours, 200 ng/mL of EGF for 10 minutes) (5). The blot was probed with Anti-Phospho-MEK1/MEK2 (Ser217, Ser221) Rabbit Monoclonal Antibody (Product # MA5-15016, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 45 kDa band corresponding to Phospho-MEK1/MEK2 (Ser217, Ser221) was detected and observed to increase upon EGF treatment across cell lines tested. Pre-treatment with EGFR-antagonist, Afatinib, resulted in inhibition of Phospho-MEK1 (Thr386) in A-431 cell line. Known quantity of protein samples were electrophoresed using Novex NuPAGE 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex Sharp Pre-Stained Protein Standard (Product # LC5800). Proteins were then transferred onto a nitrocellulose membrane with iBlot 2 Dry Blotting System (Product # IB21001). 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: FIG 4 Analysis of PGE2 biosynthesis and MEK function in THP-1 macrophages in response to treatment with DeltayopB Y. enterocolitica , YopJC172A Y. pseudotuberculosis , or the chemical MEK inhibitor PD184161. PMA-differentiated THP-1 macrophages were pretreated with or without 10 muM PD184161 (MEK1/2 inhibitor) 1 h before infection. Macrophages were uninfected (Ctrl) or were infected with wild-type or DeltayopB Y. enterocolitica (Ye) (A) or wild-type or YopJC172A Y. pseudotuberculosis (Yptb) (B and C) for 2 h at an MOI of 50:1. The PGE2 concentration from infected cell supernatants was measured by a commercial monoclonal ELISA recognizing PGE2 (A and B). Western blotting was used to quantify MEK1/2 and phosphorylated MEK/2 (C and D), where the fold change was calculated using densitometry analysis by normalizing to phosphorylated MEK 1/2 to MEK1/2 levels (D). The beta-actin was used as a loading control. Two-way ANOVA and Tukey's post hoc test were used to calculate significance for Western blot densitometry ( n = 3). P values are indicated (*, P

MA5-15016

Antibody data