Ab192234

MC3T3-E1 cells were washed in PBS and fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized with Triton X-100 (0.025%) for 10 min, blocked in 1% goat serum for 1 h and probed with primary antibody anti-Syne1 (1:100; ab192234, Abcam) at 4 °C overnight. Cells were washed and labeled with FITC-conjugated secondary antibody (ab8211, Abcam) for 1 h. Nuclei were stained with DAPI for 10 min and imaged on a confocal microscope (FV1000, Olympus, Japan).

Fibers were PFA-fixed and Triton-permeabilized as described above (Nuclear organization of single fibers). Fibers were treated with mouse-on-mouse block (Vector Laboratories, BMK-2202) for 1 hour, blocked in 10% goat serum in PBS (Sigma-Aldrich, G9023) for 30 minutes, and treated with primary antibodies diluted in goat serum blocking buffer. Prelamin A was visualized using as primary antibody Santa Cruz Biotechnology Inc. sc-6214, c-20 (secondary antibody: Alexa Fluor 488, Invitrogen, A-11078) and Pax7 using Developmental Studies Hybridoma Bank (DSHB) AB 528428 as primary antibody (secondary antibody: Alexa Fluor 488, Invitrogen, A-11001). Lamin A (Abcam, ab8980) and nesprin-1 (Abcam, ab192234) were matched with Alexa Fluor 488 and 594, respectively (Invitrogen, A-11001 and A-11012). Acetyl-histone H3 (Lys9/Lys14) antibody (Cell Signaling Technology, 9677) matched with Alexa Fluor 594, respectively (Invitrogen, A-11012).

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).