Insulinoma cells and primary islets were harvested and lysed in ice-cold RIPA buffer containing protease inhibitors (BD biosciences). Clarified cell lysates were resolved on 4–12% NuPAGE gels (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were probed with antibodies raised against the following proteins: Apaf-1 (R&D Systems Cat# MAB868), caspase 3 (Cell Signaling Technology Cat# 9662), human caspase 9 (Cell Signaling Technology Cat# 9502, RRID:AB_2068621), CHOP (Cell Signaling Technology Cat#2895), LC3B (Cell Signaling Technology Cat#2775), PARP (Cell Signaling Technology Cat# 9542, RRID:AB_2160739), XIAP (R&D Systems Cat#AF8221), and γ-tubulin (Sigma-Aldrich Cat# T6557, RRID:AB_477584). Primary antibodies were detected using goat anti-mouse IRDye 800CW (Li-COR) or Alexa Fluor 680 goat anti-rabbit IgG (Thermo Life Technologies) secondary antibodies. Immunoblots were developed using an Odyssey CLx system (Li-COR).
Apaf 1
Manufactured by R&D Systems
Sourced in United States
Apaf-1 is a protein that plays a central role in the intrinsic apoptosis pathway. It functions as an activator of caspase-9, which in turn activates other caspase enzymes, leading to programmed cell death. Apaf-1 is a key component of the apoptosome, a multiprotein complex that initiates the caspase cascade in response to cellular stress or damage.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitors, by BD (1 mentions)
Human caspase 9, by Cell Signaling Technology (1 mentions)
Odyssey clx system, by LI COR (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
γ tubulin, by Merck Group (1 mentions)
Ecl solution, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
β catenin, by BioLegend (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
3 protocols using apaf 1
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Apoptosis Markers
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Tissue lysates containing the same amount of protein (25 μg) for each experimental condition were loaded on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. Electrophoresis was run at 20 mA/gel and separated proteins were subsequently blotted on polyvinylidene fluoride membranes at 100 V for 90 minutes on ice. After overnight blocking with nonfat dry milk (5%), membranes were incubated for 1 hour with primary antibodies: APAF-1 (1 μg/mL; R&D Systems, Inc.), cathepsin B (1 μg/mL; Leica Microsystems), and β-actin (1 μg/mL; Santa Cruz Biotechnology Inc., Dallas, TX, USA). After a 1-hour incubation period in suitable secondary antibody (goat antimouse, 1:10,000 dilution; Santa Cruz Biotechnology Inc.), membranes were incubated for 3 minutes in ECL solution (Thermo Fisher Scientific, Waltham, MA, USA) and exposed to film.
3
Western Blot Analysis of Signaling Proteins
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