- PA5-19617 - Invitrogen Antibodies EEF2 antibody

Submitted references SIRT1 Protects the Heart from ER Stress-Induced Injury by Promoting eEF2K/eEF2-Dependent Autophagy.

Pires Da Silva J, Monceaux K, Guilbert A, Gressette M, Piquereau J, Novotova M, Ventura-Clapier R, Garnier A, Lemaire C
Cells 2020 Feb 12;9(2)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HEK293 whole cell lysate using Product # PA5-19617, EEF2 primary antibody at a dilution of 1 µg/mL. Blot treated with a secondary IR Dye680-conjugated Goat polyclonal anti-Rabbit antibody was used at a dilution of 1:15000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of EEF2 was achieved by transfecting HeLa with EEF2 specific siRNAs (Silencer® select Product # s4492). Western blot analysis (Fig. a) was performed using Whole cell extracts from the EEF2 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with EEF2 Polyclonal Antibody (Product # PA5-19617 at a concentration of 1 µg/mL ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 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 EEF2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-EEF2 Polyclonal Antibody (Product # PA5-19617) and a 95 kDa band corresponding to EEF2 was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), Hep G2 (Lane 2), C2C12 (Lane 3), PC-12 (Lane 4) and HeLa (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (at a concentration of 1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000 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)
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Experimental details: Immunofluorescence analysis of EEF2 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with EEF2 Polyclonal Antibody (Product # PA5-19617) at a concentration of 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 SIRT1 activates eEF2K/eEF2-dependent autophagy in response to ER stress. ( A ) H9c2 cells were treated with 10 uM A484954 for 4 h (eEF2K inhibitor) and the level of phospho-eEF2 (p-eEF2) was analyzed by Western blot (n = 7). The ratio of p-eEF2 vs. total eEF2 is presented. ( B ) H9c2 cells were left untreated or treated with 10 mug/mL tunicamycin (TN) for 48 h +- 10 uM A484954, and cell viability was determined by Fluorescein Diacetate (FDA) assay. Results presented in the bar graph are expressed as mean +- S.E.M. of percentages of dead cells (FDA negative cells, n = 11). ( C ) Autophagy was monitored by flow cytometry after Cyto-ID (r) staining of H9c2 cells untreated or treated with 10 ug/mL tunicamycin (TN) for 24 h +- 10 uM A484954 (n = 11). ( D ) Cells were left untreated or treated with 10 mug/mL tunicamycin (TN) for 24 h +- 10 uM A484954 and the levels of LC3-II were analyzed by Western blot. Actin was used as a loading control. The relative mean expression level of LC3-II is presented in the bar graph. Error bars, S.E.M. (n = 5). ( E , F , I ) H9c2 cells were transfected with control, eEF2K or eEF2 siRNA, and eEF2K ( E ) or eEF2 ( I ) expression and eEF2 phosphorylation ( F , I ) were assessed after 24 h by Western blot. ( G , J ) H9c2 cells were transfected with control, eEF2K ( G ), or eEF2 ( J ) siRNA for 24 h, then treated for 48 h with tunicamycin (TN) and cell death (FDA negative cells) was measured by flow cytometry. Data in the bar graphs represent

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 eEF2K and eEF2 are not acetylated on lysine and do not co-immunoprecipitate with SIRT1 in H9c2 cells. ( A ) Immunoprecipitation of lysine-acetylated proteins or eEF2K from lysates of H9c2 cells treated or not with 50 uM EX527 (SIRT1 inhibitor) followed by immunoblotting with anti-eEF2K or anti-acetyl lysine antibodies (n = 3). ( B ) eEF2 was immunoprecipitated from lysates of H9c2 cells treated or not with 50 uM EX527 and its level of acetylation was analyzed by immunoblotting with anti-acetyl lysine antibodies (n = 3). The location of eEF2K and eEF2 is shown by an arrow. ( C ) The physical interaction between endogenous SIRT1 and eEF2K or eEF2 was analyzed by co-immunoprecipitation. Either SIRT1 or eEF2K or eEF2 was immunoprecipitated from H9c2 lysates and the presence of SIRT1, eEF2K and eEF2 was analyzed by Western blot in each precipitate. Input: Input fraction; Output: Supernatant after immunoprecipitation; IP: Immunoprecipitate. Negative controls: IgGr: Rabbit IgG; IgGm: Mouse IgG (n = 3).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8 eIF2alpha is involved in the regulation of eEF2 phosphorylation in response to ER stress. eEF2K ( A ) and eEF2 ( B ) were immunoprecipitated from lysates of H9c2 cells treated or not with 10 mug/mL tunicamycin (TN), and the presence of eEF2K or eEF2 and eIF2alpha were analyzed by Western blot (n = 3). ( C , D ) H9c2 cells were transfected with control or eIF2alpha siRNA for 24 h, treated with tunicamycin (TN) for 1h30, and the level of eIF2alpha or phospho-eEF2 was analyzed by Western blot (RD: Relative density). The ratio of p-eEF2 vs. total eEF2 is presented in the bar graph. Error bars, S.E.M. (n = 4). ** P < 0.01 versus CTL siRNA+TN.

PA5-19617

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