Protein extracts were prepared from frontal and parietal bones of one-month-old mice after cutting out the sutures. The bone tissues were immersed into liquid nitrogen, grinded to powder, and then lysed using NP40 lysis buffer (PH 8.0 Tris-HCl 20mM, NaCl 137mM, 1% NP40, 10% Glycerol, Na3VO4 1mM). Western blotting was performed and analyzed similar to our previous studies [39 (link)–41 (link)]. The following primary antibodies were used: rabbit anti-RBCC1 (FIP200) antibody (proteintech, 17250-1-AP), rabbit anti-P62 antibody (Enzolife science BML-PW9860-0100), mouse anti-LC3 antibody (Nanotools, NT-0231-100), mouse anti-vinculin antibody (Sigma-aldrich V4505), and rabbit anti-ATG5 antibody (CST, #13007).
Protocol detail
Bone Protein Extraction and Analysis
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Anti antibody
Antibodies
Antibody
Bone tissues
Buffer
Glycerol
Mouse
Nacl
Nitrogen
Parietal bones
Powder
Protein
Rabbit
Sutures
Tris
Vinculin
Corresponding Organization :
Other organizations : University of Michigan–Ann Arbor, University of Cincinnati Medical Center
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Variable analysis
independent variables
- Location of bone tissue (frontal vs. parietal)
- Protein expression levels of RBCC1 (FIP200), P62, LC3, and ATG5
- Age of mice (one-month-old)
- Tissue preparation method (immersion in liquid nitrogen, grinding to powder, lysis with NP40 buffer)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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