Anti ucp2

Mouse NB cell line Neuro-2a, human NB cell line BE(2)-M17, and SH-SY5Y were purchased from the Cell Center of the Chinese Academy of Medical Science. Neuro-2a, BE(2)-M17, and SH-SY5Y were cultured in MEM, DMEM, and DME/F12 (HyClone, Logan, UT, USA) medium containing 10% FBS (Gibco Grand Island, NY, USA), respectively, and grown in a 37°C incubator with 5% CO₂. Genipin or T0090709 was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Anti-UCP1, anti-N-Cadherin, anti-Vimentin, anti- E-Cadherin, anti-UCP2, anti-CDK4, anti-cyclin D1, and anti-PPARγ antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA), and secondary antibody and anti-β-actin antibodies were purchased from (Proteintech, Wuhan, China). Other chemical reagents were provided by Sigma (Sigma-Aldrich, St. Louis, MO, USA).

HRECs were lysed in cell lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1-mM phenylmethylsulfonyl fluoride and a protease/phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA). Total protein concentrations were measured using a bicinchoninic acid assay (Beyotime Biotechnology). Protein samples (30–50 µg) were used for western blot using 4% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to a 0.22-µm polyvinylidene fluoride membrane (Roche). After being blocked in nonfat milk, the membranes were incubated in the primary antibody at 4°C overnight and in the secondary antibody (The Jackson Laboratory, West Grove, PA, USA) at room temperature for 1 hour. The ECL Plus HRP substrate (EMD Millipore, Burlington, MA, USA) and Amersham Imager 600 (General Electric, Boston, MA, USA) were used to visualize the immunoreactive bands. The primary antibodies were as follows: anti-UCP2 (#89326), anti-SIRT3 (#2627), anti-SOD2 (#13141), anti-P21 (#2947), and anti-Actin (#5125), all of which were purchased from Cell Signaling Technology (Danver, MA, USA), and Anti-beta Tubulin antibody (#ab6046), which was purchased from Abcam (Cambridge, UK).