NK-4 (5 μM)-treated THP-1 macrophages were incubated with or without Apo-J cells at a ratio of 1:1 for 45 min at 37 °C. Whole-cell extracts were prepared with RIPA buffer (Wako Pure Chemical) containing phosphatase inhibitor (Nacalai Tesque Inc., Kyoto, Japan) and protease inhibitor (Roche Diagnostics, Mannheim, Germany) and subjected to western immunoblotting. Membranes were probed with a 1:1000 dilution of anti-phospho-Akt (Ser473) rabbit pAb (9171; Cell Signaling Technology, Danvers, MA). Specific bands were detected using an ECL™ Plus Western Blotting System (Immobilon Western Chemiluminescent HRP substrate; GE Healthcare, UK). After treatment with a reprobing solution (Restore Western Blot Stripping Buffer; Pierce Biotechnology, Rockford, IL) for 15 min at room temperature, the membrane was used for secondary detection with a 1:1000 dilution of anti-Akt rabbit mAb (C67E7; Cell Signaling Technology). Band density was measured using ImageQuant TL software (GE Healthcare).
Ecl plus western blotting system immobilon western chemiluminescent hrp substrate
Manufactured by GE Healthcare
Sourced in United Kingdom
The ECL™ Plus Western Blotting System is a chemiluminescent HRP substrate developed by GE Healthcare. It is designed for the detection of proteins in Western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Phosphatase inhibitor, by Nacalai Tesque (2 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Imagequant tl software, by GE Healthcare (2 mentions)
Anti phospho akt ser473 rabbit pab 9171, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Fujifilm (2 mentions)
2 protocols using ecl plus western blotting system immobilon western chemiluminescent hrp substrate
1
Evaluating Akt Phosphorylation in THP-1 Macrophages
2
Akt Phosphorylation in Macrophage-Apo-J Interaction
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(5 mM)-treated THP-1 macrophages were incubated with or without Apo-J cells at a ratio of 1:1 for 45 min at 37°C. Whole-cell extracts were prepared with RIPA buffer (Wako Pure Chemical) containing phosphatase inhibitor (Nacalai Tesque Inc., Kyoto, Japan) and protease inhibitor (Roche Diagnostics, Mannheim, Germany) and subjected to western immunoblotting. Membranes were probed with a 1:1000 dilution of anti-phospho-Akt (Ser473) rabbit pAb (9171; Cell Signaling Technology, Danvers, MA). Speci c bands were detected using an ECL ™ Plus Western Blotting System (Immobilon Western Chemiluminescent HRP substrate; GE Healthcare, UK). After treatment with a reprobing solution (Restore Western Blot Stripping Buffer; Pierce Biotechnology, Rockford, IL) for 15 min at room temperature, the membrane was used for secondary detection with a 1:1000 dilution of anti-Akt rabbit mAb (C67E7; Cell Signaling Technology). Band density was measured using ImageQuant TL software (GE Healthcare).
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