Radioimmunoprecipitation assay (RIPA) buffer was used to obtain cell lysates from both SH-SY5Y cells, and SNpc region of the brain. Protein concentrations were determined, using the Bradford reagent (Bio-Rad), and 25 ug of lysates were resolved on NuPAGE 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA) followed by Western blotting as described [25 (link), 26 (link)], using the following monoclonal or polyclonal antibodies: anti-TRPM7 (Abcam, MA; Cat# 109438; Dilution in 1:500), anti-LC3A (Cell Signaling, MA; Cat# 4599; Dilution in 1:1000), anti-Bcl2 (Cell Signaling, MA; Cat# 209039; Dilution 1:1000), anti-Bax (Cell Signaling, MA; Cat# 5023; Dilution used was 1:1000), anti-Caspase3 (Cell Signaling, MA; Cat# 9662; Dilution used was 1:2000), anti-β-Actin (Cell Signaling, MA; Cat# 4970; at 1:2000 dilution)
Protocol detail
Western Blot Analysis of Autophagy and Apoptosis Markers
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Bradford reagent
NuPAGE 4–12% Bis-Tris gels
anti-TRPM7
anti-LC3A
anti-Bcl2
anti-Bax
anti-Caspase3
anti-β-Actin
Actin
Antibodies
Bcl2
Bis tris
Brain
Buffer
Caspase3
Cells
Dilution
Gels
Protein
Radioimmunoprecipitation assay
Trpm7
Corresponding Organization :
Other organizations : The University of Texas Health Science Center at San Antonio
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Variable analysis
independent variables
- Not explicitly mentioned
- TRPM7 protein levels
- LC3A protein levels
- Bcl2 protein levels
- Bax protein levels
- Caspase3 protein levels
- Protein concentrations determined using Bradford reagent
- Equal amounts of lysates (25 μg) resolved on NuPAGE 4–12% Bis-Tris gels
- β-Actin protein levels used as loading control
- Positive control: Not mentioned
- Negative control: Not mentioned
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