- 700256 - Invitrogen Antibodies AKT1 antibody

Franco C, Kausar S, Silva MFB, Guedes RC, Falcao AO, Brito MA
Cancers 2022 Jul 19;14(14)

Ahmad F, Salahuddin M, Alsamman K, Herzallah HK, Al-Otaibi ST
Bioscience reports 2018 Jun 29;38(3)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of AKT (pS473) was performed by loading 20 µg of NIH/3T3 (lane1), NIH/3T3 treated for 10 minutes with 25 ng/mL of PDGF (lane2) and U-87 MG (lane3) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. AKT (pS473) was detected at ~55 kDa using AKT (pS473) Recombinant Rabbit Monoclonal Antibody (Product # 700256) at 0.5 µg-1 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in the corresponding blot on right. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of AKT (pS473) was done on 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with AKT (pS473) Recombinant Rabbit Monoclonal Antibody (Product # 700256) at 1 µg-2 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization of AKT (pS473). Panel e shows competition with AKT (pS473) peptide. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of AKT [pS473] was done on U-87 MG cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª AKT [pS473] Recombinant Rabbit Monoclonal Antibody (700256, red histogram) or with rabbit isotype control (pink histogram) at 2 µg-4 µg/million cells in 2.5% BSA. After incubation at room temperature for 2-3 hours, the cells were labeled with Alexa Fluor¨ 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 PI3Kp110beta inhibitor candidates lead to decreased phospho-AKT expression and increased glioma cell apoptosis. U87MG cells were grown on coverslips for 48 h and then incubated with molecule (Mol) 19 or 20 at EC 50 , or vehicle (control; Ctr): ( A ) Immunostaining for phospho-AKT and nuclei labeling with Hoechst 33342 were performed at different time-points. Images are representative of three independent experiments each with 10 random fields analyzed. ( B ) Semi-quantitative analysis of phospho-AKT expression along time per cell. ( C ) Analysis of the percentage of apoptotic cells. White arrows in ( A ) point to cells presenting characteristic morphological changes of apoptosis such as condensation of chromatin and nuclear fragmentations, whereas red arrows point to necrotic-like areas with consistent nuclear lysis. Data presented in ( B , C ) are means +- SEM of three independent experiments, each with 10 random fields analyzed. Statistical analysis was performed by one-way ANOVA with Tukey correction. * p < 0.05, *** p < 0.001 vs. control of each time-point; # p < 0.05, ## p < 0.01, ### p < 0.001 between indicated groups.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Dual targeting candidates lead to decreased phospho-EGFR and -AKT expression and increased glioma cell apoptosis. U87MG cells were grown on coverslips for 48 h and then incubated with molecule (Mol) 25 or 27 at EC 50 , or vehicle (control; Ctr): ( A ) Double immunostaining for phospho-EGFR and phospho-AKT were performed at different time points. Nuclei were counterstained with Hoechst 33342. Images are representative of three independent experiments each with 10 random fields analyzed: ( B ) Semi-quantitative analysis of phospho-EGFR expression along time per cell. ( C ) Semi-quantitative analysis of phospho-AKT expression along time per cell. ( D ) Analysis of the percentage of apoptotic cells. White arrows in ( A ) point to cells presenting characteristic morphological changes of apoptosis such as condensation of chromatin and nuclear fragmentations, whereas red arrows point to necrotic-like areas with consistent nuclear lysis. Data presented in ( B - D ) are means +- SEM of three independent experiments, each with 10 random fields analyzed. Statistical analysis was performed by one-way ANOVA with Tukey correction. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. control of each time-point; # p < 0.05, ## p < 0.01, ### p < 0.001 between indicated groups.

700256