Ab124916

Cells were lysed with radio-immunoprecipitation assay lysis buffer (APExBIO, K1020), supplemented with protease inhibitor cocktail (APExBIO, K1007) and phosphatase inhibitor cocktail (APExBIO, K1015). Insoluble cell debris was removed by 15-min centrifugation at 13,500×g at 4 °C. Protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime, P0010S). Western blot was performed on total protein extracts. Proteins were then transferred to 0.45-μm polyvinylidene difluoride membranes (Sigma, IPVH00010) after electrophoresis. Membranes were blocked for 1 h at room temperature in blocking buffer (Beyotime, P0023B) and incubated overnight at 4 °C with antibodies in primary antibody dilution buffer (Beyotime, P0023A). Goat-anti-Rabbit horseradish peroxidase-conjugated (Abcam, ab2057181:10,000) antibody was used as secondary antibody. Protein bands were visualized with Clarity Western ECL Substrate (Bio-Rad, USA) and the pictures were obtained by ChemiScope Western Blot Imaging System (Clinx Science Instruments, China).
Primary antibodies and corresponding concentrations were used as follows: rabbit monoclonal anti-SUN1 (Abcam, ab124770, 1:1000) and rabbit monoclonal anti-SUN2 (Abcam, ab124916, 1:1000). Glyceraldehyde 3-phosphate dehydrogenase (Abcam, ab8245, 1:1000) was used as a loading control.

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).

Western-blot analysis was performed according to standard protocols as described previously [11 (link)]. In brief, membranes (PVDF membranes) were incubated with antibodies against Sun2 (ab124916, Abcam, UK), α-tublin (H3663, Sigma Aldrich, USA) or SAA1 (ab201660, Abcam, USA). The intensities of light-emitting bands were checked and quantified using AmershamImager 600 (GE Healthcare,USA). α-tublin was used as an internal control.