Cell lysis, SDS‐PAGE and immunoblot analysis were performed as described previously.8 (link) Membranes were incubated with antibodies against the following proteins: BIG3 (diluted 1:250),6 (link) PHB2 (Abcam #ab71970, diluted 1:1000), β‐actin (clone AC‐15, Sigma‐Aldrich #A1978, diluted 1:5000), PARP (CST #9542, diluted 1:500), and apoptosis‐inducing factor (AIF) (clone E‐1, Santa Cruz Biotechnology #sc‐13116, diluted 1:200). After incubation with HRP‐conjugated secondary antibodies (anti‐mouse IgG‐HRP, diluted 1:5000; anti‐rat IgG‐HRP, diluted 1:5000; anti‐rabbit IgG‐HRP, diluted 1:1000 [Santa Cruz Biotechnology]) for 1 hour, the blots were developed with an enhanced chemiluminescence system (GE Healthcare) and scanned using an Image Reader LAS‐3000 Mini (Fujifilm).
Protocol detail
Cell Lysis and Immunoblot Analysis
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β‐actin (clone AC‐15,
anti‐mouse IgG‐HRP
anti‐rat IgG‐HRP
anti‐rabbit IgG‐HRP
enhanced chemiluminescence system
Image Reader LAS‐3000 Mini
Actin
Anti igg
Antibodies
Apoptosis inducing factor
Cell
Chemiluminescence
Clone
Immunoblot analysis
Membranes
Mouse
Phb2
Proteins
Rabbit
Sds page
Corresponding Organization : Tokushima University
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Variable analysis
independent variables
- None explicitly mentioned
- Protein expression levels of BIG3, PHB2, β-actin, PARP, and apoptosis-inducing factor (AIF)
- None explicitly mentioned
- Positive control: None mentioned
- Negative control: None mentioned
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