- MA5-15116 - Invitrogen Antibodies MAPK14 antibody

Submitted references p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.

Loonat AA, Martin ED, Sarafraz-Shekary N, Tilgner K, Hertz NT, Levin R, Shokat KM, Burlingame AL, Arabacilar P, Uddin S, Thomas M, Marber MS, Clark JE
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2019 Dec;33(12):13131-13144

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing ofp38α MAPK14 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR611649_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of p38α MAPK14 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with p38α MAPK14 Lentiviral sgRNA (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-p38 MAPK alpha Monoclonal Antibody (J.491.1) (Product # MA5-15116) using 1:1,000 dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177 1:6,000 dilution).Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific top38α MAPK14 (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of p38 MAPK alpha was achieved by transfecting HeLa with p38 MAPK alpha specific siRNAs (Silencer® select Product # s3586, s3587). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the p38 MAPK alpha knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with p38 MAPK alpha Monoclonal Antibody (J.491.1) (Product # MA5-15116, 1:1000 dilution) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP, 1:6000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to p38 MAPK alpha.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-p38 MAPK alpha Monoclonal Antibody (J.491.1) (Product # MA5-15116) and a 38kDa band corresponding to p38 MAPK alpha was observed across cell lines tested. Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), Jurkat (Lane 2), A549 (Lane 3), A-431 (Lane 4), MCF7 (Lane 5), Neuro-2a (Lane 6) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:6000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of MAPK14 was performed with 10 µg of A549 cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against MAPK14 (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. MAPK14 was detected at ~ 43 kDa using MAPK14 Rabbit polyclonal antibody (Product # MA5-15116) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Rabbit (Product # 31461) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to Cyclophilin B (21 kDa). MAPK14 Antibody (Product # MA5-15116) confirms silencing of MAPK14 expression.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of p38 MAPK alpha using a monoclonal antibody (Product # MA5-15116).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of p38 MAPK alpha using a monoclonal antibody (Product # MA5-15116).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of p38 MAPK alpha using a monoclonal antibody (Product # MA5-15116).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of p38 MAPK alpha using a monoclonal antibody (Product # MA5-15116).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

MA5-15116

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