Anti lamin a c antibody

Nuclear fractionation was carried out as previously described by Xu et al.^{28 (link)} Cells were harvested after 24 h, and the cytoplasmic and nuclear fractions were separated and extracted by using a NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo-Fisher Scientific). SerRS was detected with an anti-SerRS antibody (made in our lab). An anti-Lamin A/C antibody (Cell Signaling Technology, Danvers, MA, USA) and an α-tubulin antibody (Proteintech, China) were used to identify nuclear and cytoplasmic markers, respectively.

Insulin, dexamethasone (Dex), 3-isobutyl-1-methylxanthine (IBMX), Hoechst 33258, and Oil Red-O were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-lamin A/C antibody was from Cell Signaling Technology (Danvers, MA, USA), anti-FLAG (900013) antibody from Gene Script (Piscataway, NJ, USA), anti-YAP antibody (SC101199) was from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-β-actin (AC-15) was from Sigma-Aldrich (St. Louis, MO, USA). Anti-Wnt5a (MAB645) and anti-14-3-3γ (MAB5700) antibodies were obtained from R&D Systems (Minneapolis, MN, USA) and BODIPY493/503 (D3922) and fluorescent phalloidin conjugate (A34055) from Invitrogen (Waltham, MA, USA). cDNAs of Flag-YAP-5SA were subcloned into the pBABE retroviral vector. Normal diet (ND) (3.28 kcal/ g containing 14% of calories from fat) was purchased from Altromin Spezialfutter GmbH & Co. KG. (Lage, Germany) and the high-fat diet (HFD) (5.24 kcal/g containing 60% of calories from fat) from Research Diets, Inc. (New Brunswick, NJ, USA). Recombinant human IL‐6, IL-1β, and TNF-α were purchased from Peprotech (Saint Paul, MN, USA), and ELISA kits (OKEH04420) from AVIVA (San Diego, CA, USA).

Western blot analyses were performed as described previously 15. Primary antibodies used for western blot and dilution are as follows: anti‐FLAG M2–horse radish peroxidase (HRP) antibody (Sigma‐Aldrich, 1 : 1000), anti‐myc–HRP antibody (Thermo Fisher Scientific, 1 : 5000) and anti‐V5–HRP antibody (Thermo Fisher Scientific, 1 : 5000), anti‐Lamin A/C antibody (Cell Signaling Technology, Danvers, MA, USA, 1 : 1000), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (Cell Signaling Technology, 1 : 500), anti‐p27^kip1 antibody (MBL, 1 : 1000), anti‐JAB1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, 1 : 200), anti‐Cdk2 antibody (Santa Cruz Biotechnology, 1 : 200), and anti‐cyclin E antibody (MBL, 1 : 1000). Anti‐mouse IgG HRP‐linked and anti‐rabbit IgG HRP‐linked (Cell Signaling Technology, 1 : 5000) secondary antibodies were used. Data were analyzed using lumivision analyzer 400 software (Aisin Seiki, Kariya, Aichi, Japan).