Lamin a c antibody

Cells were lysed in ice cold RIPA buffer, incubated for 30 min in 4 °C and centrifuged at 14,400× g for 20 min at 4 °C. Samples were resolved using SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (PVDF) (Thermo Fisher Scientific). USP8 protein was detected with USP8 Rabbit polyclonal antibody (27791-1-AP, Proteintech, Rosemont, IL, USA), p27 protein was detected with Anti-p27 KIP1 antibody (ab32034, Abcam, Waltham, MA, USA). Reference proteins Lamin A/C and Glyceraldehyde-3-Phosphate Dehydrogenase were detected with Lamin A/C antibody (sc-20681, Santa Cruz Biotechnology, Dallas, TX, USA) and Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (#MAB374, Sigma-Aldrich), respectively. Anti-rabbit antibody conjugated to HRP (#7074, Cell Signalling, Danvers, MA, USA) was used as secondary antibody. SuperSignal West Pico Chemiluminescent Substrate (no. 34087, Thermo Fisher Scientific) and the CCD digital imaging system Alliance Mini HD4 (UVItec Limited, Cambridge, United Kingdom) were used for visualization. Densitometry was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

pGCTB-SCs were seeded in 6-well dishes at a 1.2 × 10⁶ cells/well density and incubated overnight. The following day, the culture media were replaced for each reagent, and the cells were incubated for an additional 12 h. After incubation, the cells were washed twice with ice-cold PBS, scraped, and centrifuged. Cytoplasmic and nuclear proteins were isolated using nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific), to which Cell Lytic M (Sigma-Aldrich) with protease inhibitor cocktail (cOMplete™ Mini: Sigma-Aldrich) were added.
Western blotting was performed as previously described ^{45 (link)} with the following primary antibodies: β-catenin (1:1000) and actin clone C4 (1:5000), with or without rabbit polyclonal Lamin A/C antibody (1:3000, sc-20681; Santa Cruz Biotechnology). Relative intensity was calculated using the ratio of each target protein's signal intensity to internal controls' intensity, using ImageJ ver1.52p (NIH, Bethesda, MD, USA). At least three separate experiments were conducted. Full-length blots were absent because membranes were cut prior to hybridization with primary antibodies. The unedited blots including replicates were shown in Supplementary Figs. S5, S6, S7.

Lamin A/C antibody (Santa Cruz, sc376248) was used at 1:200 at 4 °C overnight.