Ab226198

Whole-cell lysates were harvested with 200 μl of RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific). Cytoplasmic and nuclear extracts were prepared with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Western blots were performed by standard procedures using antibodies for Smad1 (1:1000 dilution, #9743, Cell Signaling Technology), phospho-Smad1/5/9 (1:1000 dilution, #13820, Cell Signaling Technology), β-actin-HRP (1:10,000 dilution, PM053-7, MBL, Japan), Lamin A (1:1000 dilution, ab226198, Abcam), β-tubulin (1:1000 dilution, H-235, Santa Cruz), rabbit IgG–peroxidase (1:20,000 dilution, A0545, Sigma-Aldrich), and S-protein HRP conjugate (1:5000 dilution, 69047, Novagen). The signals were detected by ECL Prime Western Blotting Detection Reagents (RPN2232, GE Healthcare). Quantification was performed using ImageJ software.

Co‐immunoprecipitation was performed as described previously.²⁷, ²⁸, ³⁰, ³¹ Cell lysates were mixed with protein A/G‐magnetic beads (Novex, Oslo, Norway) and incubated at 4°C overnight with the selected antibodies. The beads were washed using Western/IP lysis buffer (Beyotime, Haimen, China, composition: 20 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X‐100, and inhibitors containing sodium pyrophosphate, β‐glycerophosphate, EDTA, Na₃VO₄ and leupeptin), and eluted using the Acid Elution buffer (Beyotime). Next, total protein amount of each group was measured by BCA protein quantification. After the first quantification, the samples were diluted according to the difference in protein amount, and the second quantification was carried out for confirmation. Finally, the samples were suspended in SDS‐PAGE loading buffer and then measured by IB. The antibodies used for co‐IP were as follows: anti‐FLAG (CST, #14793 and #8146), anti‐PDHA (Abcam, #ab168379 and #ab110330), anti‐HA (Abcam, #ab9110 and #ab1424), anti‐EMD (Abcam, #ab40688 and #ab204987), anti‐Lamin A (Abcam, #ab226198), anti‐Nesprin1 (Abcam, #ab192234), anti‐Nesprin3 (Abcam, #ab186746), anti‐Sun1 (Abcam, #ab124770) and anti‐Sun2 (Abcam, #ab124916).