Ab18554

The samples were mixed with 2×SDS lysis buffer (50 mM Tris–HCl at pH 6.8, 2% SDS and 10% glycerol) with 1×Protein Inhibitor Cocktail, boiled at 100 °C for 10 min. The supernatant was obtained by centrifugation at 13,000 g for 10 min and the protein concentration was determined using the Pierce^TM BCA protein Assay kit (Thermo Fisher). Proteins in lysates were separated by SDS-PAGE, transferred to PVDF membranes, and blocked in 5% skim milk that contained 0.1% Tween 20 at room temperature for 1 h. The membranes were immunoblotted with the corresponding antibodies overnight at 4 °C, and then were washed and incubated with horseradish peroxidase conjugate secondary antibodies at room temperature for 1 h. After washing, the bands were visualized using the Pierce™ ECL Western Blotting Substrate kit (Thermo Fisher) and band intensities were quantified by ImageJ. The antibodies were listed as following: CNTNAP2 (ab33994, Abcam), Necdin (ab18554, Abcam), Flag-tag (14793, CST), HA-tag (3724, CST), GFP (2555, CST), CASK (ab252540, Abcam), GAPDH (97166, CST), Histone H3(9715, CST), PS1 (ab15458, Abcam).

Co-immunoprecipitation experiments on nuclear extracts from WT and mdx TA muscles were performed by following the previously described protocol with modifications [29 (link)]. Briefly, 1 mg of proteins were incubated with 10 μg of p75NTR antibody (Sigma-Aldrich, Milan, Italy; 07-476). Nuclear fractions were probed with the antibody for 1 h at 4 °C, subsequently 50 μL of protein A-agarose was added for 1 h at 4 °C. Samples were then centrifuged (50,000× g for 15 min), and the pellet was gently washed three times with washing buffer to eliminate non-specific binding. Immunoprecipitated proteins were denatured in Laemmli buffer by heat treatment and separated in SDS-PAGE. Proteins were electrophoretically transferred onto nitrocellulose and then probed overnight at 4 °C with anti-p75NTR (Santa Cruz Biotechnology, sc-271708, dilution 1:1000) or anti-Necdin (Abcam, ab18554, dilution 1:1000) antibodies. For necdin visualization, secondary anti-rabbit IgG HRP “True blot” (Rockland Immunochemicals) was employed.

Antibodies against Necdin (ab18554), SGT1 (ab30931), BMAL1 (ab3350), HSP90 (ab13492), PER1 (ab136451), PER2 (ab180655), CRY1 (ab104736) were purchased from Abcam. Antibody against AVP (sc-390723) were obtained from Santa Cruz. Antibodies against Myc-tag (2276), Flag-tag (14793), HA-tag (3724), VIP (63269) were obtained from Cell Signaling Technology. Antibody against CRY2 were obtained from Invitrogen. Antibody against GRP (20073) were obtained from ImmunoStar. 17-AAG (HY-10211) and Geldanamycin (HY-15230) were purchased from MedChemExpress.