Anti lamin b

To confirm the clean separation of the nuclear and cytoplasmic components, the respective extracts were assessed for protein presence and concentration using the DC Protein Assay Kit (Bio-Rad, Cat#: 5000111, Berkeley, CA, USA). Western blots were performed as previously described [10 (link)], and CIPGC samples were used as a positive control. Total protein was lysed using RIPA buffer to extract nuclear, cytoplasmic, and cellular components, respectively. The samples were electrophoresed on 11% sodium-dodecyl sulfate polyacrylamide (SDS-PAGE) gel. The primary antibodies anti-Lamin B (1:10,000 dilution, Abcam, Cat#: ab16048 Cambridge, MA, USA) and anti-GAPDH (1:5000 dilution, Santa Cruz, Cat#: sc-137179, Dallas, TX, USA) were used to test the purity of both the cytoplasmic and nuclear samples, respectively. Anti-mouse IgG (1:5000 dilution, HRP-linked; Cell Signaling Technology; Cat#: 7076, Danvers, MA, USA) was used to detect anti-GAPDH, and anti-rabbit IgG (1:10,000 dilution, HRP-linked; Cell Signaling Technology; Cat#: 7074S, Danvers, MA, USA) was used to detect anti-Lamin B. Anti-FOXO3 (Abcam, Cat#: ab12162, Cambridge, UK) primary antibody was used to test the expression level of FOXO3. The membrane was imaged using Image Lab Software (Version 6.1, Bio-Rad, Berkeley, CA, USA). Each experiment was performed in triplicate.