Lamin a c

Cell fractionation assays were performed using Nuclear and Cytoplasmic Extraction Reagents (Pioneer Biotechnology, Xi’an, China) following the manufacturer’s instructions. The co-immunoprecipitation (Co-IP) assay were conducted by a Dynabeads protein G immunoprecipitation kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Equal amounts of protein were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were blocked in 5% nonfat milk and then incubated overnight at 4 °C with the primary antibodies against FMNL2, MCM7, PCNA and p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Ki67 and ERα (Abcam, Cambridge, MA, USA); Ubiquitin and PR (Cell Signaling Technology, USA.); HER2, p21, CyclinD1, Lamin A/C, GAPDH and β-actin (Proteintech, Wuhan, China). After incubation with proper HRP-conjugated secondary antibody, the protein bands were visualized by an enhanced chemiluminescence system (Millipore, Bedford, MA, USA).

CEP (Cepharanthine, purity > 98%, MB2077-S) was purchased from Meilunbio (Dalian, China). CHX (Cycloheximide, S7418), MG132 (S2619) and ActD (Actinomycin D, S8964) were obtained from Selleck (Houston, TX, USA). Promega (Madison, WI, USA) provided the product of dual-luciferase reporter assay kits (E1960). Invitrogen (Camarillo, CA, USA) supplied the product of Lipofectamine^® 3000 reagent (L3000015).
The antibodies used in the present investigation are as follows: β-catenin (Proteintech, 51067-2-AP), Axin2 (Abclonal, A17022), CyclinD1 (Proteintech, 60186-1-Ig), β-actin (Sigma-Aldrich, A1978), Lamin A/C (Proteintech, 10298-1-AP), Phospho-β-catenin (Cell Signaling Technology, 9561), Active-β-catenin (Cell Signaling Technology, 4270).

T0901317, bexarotene, ABC294640, SLM6031434, and FTY720 were purchased from Cayman Chemical (Ann Arbor, MI). Trichostatin A (#HY-15144) was purchased from MedChemExpress (Monmouth Junction, NJ). Synthetic Aβ_1-42 peptide (Peptide Institute, Osaka, Japan) was solubilized in hexafluoro-2-propanol at a concentration of 1 mg/ml to prevent self-aggregation. For immunoblotting and immunostaining, antibodies against human Aβ N terminal (clone 82E1; Immuno-Biological Laboratories, Gunma, Japan), ATP binding cassette transporter A1 (ABCA1) (clone HJ1; Abnova, Taipei, Taiwan), ApoE (#AB947; Merck Millipore, Burlington, MA), α-tubulin (clone 10G10; Fujifilm Wako, Osaka, Japan), β-actin (#5125; Cell Signaling Technology, Danvers, MA), GAPDH (#2118; Cell Signaling Technology), glial fibrillary acidic protein (GFAP) (#GTX108711; GeneTex, Irvine, CA), Lamin A/C (#10298-1-AP; Proteintech, Rosemont, IL), LXRα/β (#sc-377260; Santa Cruz Biotechnology, Dallas, TX), nuclear factor-κB (NF-κB) p65 (#8242; Cell Signaling Technology), RXRα (#sc-515929; Santa Cruz Biotechnology), SphK2 (#17096-1-AP; Proteintech), and 6His (#66005-1-Ig; Proteintech) were purchased from the indicated vendors.

Mouse colon tissues or RAW264.7 cells were added with an appropriate amount of RIPA buffer, followed by protease inhibitors and phosphatase inhibitors, and lysed on ice for 40 min. Then, nuclear and cytoplasmic proteins from RAW264.7 cells were separated using the Nuclear and Cytoplasmic Extraction Kit. After protein quantification, the same amount of protein was added to each gel well, and separated by 10% resolving gel and 5% stacking gel and transferred onto PVDF membranes. After blocking with 5% BSA for 2–3 h, incubated PVDF membranes with NOS2, Arg-1 (rabbit polyclonal, 1:500), Lamin A/C (rabbit polyclonal, 1:1,000) (all purchased from Proteintech, Chicago, IL, United States); and p-STAT1, STAT1, p-STAT6, and STAT6 (rabbit polyclonal, 1:500) (all from Affinity, Shanghai, China); ß-actin (rabbit polyclonal, 1:1,000) (from Santa Cruz, Dallas, TX, United States) at 4°C overnight. Next, the membranes were disposed with HRP-labeled Goat IgG antibody (anti-rabbit, 1:10,000) (from Zhong Shan, Beijing, China) for 1 h. Then the membranes were washed with 1×PBST, and incubated in a photoluminescence solution for imaging. The bands of the protein were analyzed by Image J software (version: v1.8.0).

Total proteins were prepared using prechilled RIPA buffer (Thermo Fisher, USA) with proteinase inhibitor cocktail (Thermo Fisher, USA). For separating nuclear and cytoplasmic proteins, cells were harvested and lysed in nuclear and cytoplasmic extraction reagents (Keygentec, China) according to the manufacturer’s protocols. The proteins were subjected to western blot using antibodies against E2F1 (1:1000, Cell Signaling, USA), N-cadherin (1:1000, Proteintech, USA), MMP2 (1:1000, Proteintech, USA), MMP9 (1:1000, Proteintech, USA), Snail1 (1:1000, ABclonal, China), GAPDH (1:5000, Proteintech, USA), Lamin A/C (1:1000, Proteintech, USA). Relative expression levels were normalized to endogenous loading control using ImageJ software (National Institutes of Health, USA).

Melanoma cells were seeded at a density of 6 × 10⁵ cells ml⁻¹ in 12-well plates and lysed the next day with LDS buffer 1× (Life Technologies). Lysates were denatured at 95 °C for 5 min and sonicated. SDS–PAGE and western blotting were performed using standard procedures. Membranes were visualized and bands quantified using an Odyssey Fc (LI-COR). Loading controls were run on the same blot. Individual 680 and 800 channels were co-visualized in greyscale on the same image. Primary antibodies were: LAP1 (1:1,000; #21459-1-AP), Lamin A/C (1:1,000, #10298-1-AP), Lamin B1 (1:1,000, #12987-1-AP), Lamin B2 (1:1,000, #10895-1-AP) from Proteintech; pThr18/Ser19-MLC2 (1:750, #3674), MLC2 (1:750, #3672) from Cell Signaling Technology; GAPDH (1:10,000, #MAB374) from Merck. Secondary antibodies were: IRDye 680RD goat anti-rabbit IgG (1:10,000, #925-68071) and IRDye 800RD goat anti-mouse IgG (1:10,000, #925-32210) from LI-COR.

Equal quantities of the proteins were separated by SDS-PAGE gels, transferred to nitrocellulose membranes, incubated with 5% skimmed milk, and then incubated with the primary antibodies at 4 °C overnight. The primary antibodies were diluted as follows: NRF2 (1:1000, GeneTex, Irvine, CA, USA), β-tubulin (1:2000, ZenBio, Chengdu, Sichuan, China), and Lamin A/C (1:2000, Proteintech, Chicago, IL, USA). The membranes were incubated with the HRP-labeled secondary antibody in blocking buffer at room temperature. Blots were developed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA). The relative protein density was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).