Western Blot Analysis of NTF2 and Lamins

Whole cell lysates were prepared using SDS-PAGE sample buffer supplemented with benzonase nuclease (Sigma, E1014) and boiled for 5 min. Proteins were separated on SDS-PAGE gels (4–20% gradient) and transferred to PVDF membrane. Membranes were blocked in Odyssey PBS Blocking Buffer (Li-Cor, 927–40,000). The primary antibodies used were mouse anti-NTF2 at 1:500 (Sigma, N9527), rabbit anti-NTF2 at 1:200 (Aviva System Biology, ARP64840_P050), mouse anti-lamin A/C at 1:500 (Santa Cruz Biotech sc-376248), rabbit anti-lamin B1 at 1:2000 (Abcam 16,048), and mouse anti-actin at 1:200 (Abgent, AM1965b). The secondary antibodies were IRDye 680RD anti-mouse used at 1:20,000 (Li-Cor 925-68070) and IRDye 800CW anti-rabbit used at 1:20,000 (Li-Cor 925-32211). Blots were scanned on a Li-Cor Odyssey CLx instrument and band quantification was performed with ImageStudio. For a given sample, NTF2 band intensity was normalized to the actin signal, and lamin band intensity was normalized to Ponceau-stained total protein. For western blots on LNCaP cell lysates, HRP detection was used as previously described^{73 (link)}.