3 isobutyl 1 methylxanthine ibmx i5879

The following antibodies were used in this study for western blots and immunoprecipitation assays: anti-CHIP (rabbit C3B6; Cell Signaling Technology, Danvers, Massachusetts, USA), MYC (sc-40; Santa Cruz Biotechnology, CA, USA), FLAG (mouse F3165; Sigma-Aldrich, St. Louis, MO, USA), FLAG (rabbit; Sigma-Aldrich), HA (mouse sc-7392; Santa Cruz Biotechnology, CA, USA), PPARγ (mouse sc-7273X, Santa Cruz Biotechnology), PPARγ (rabbit sc-7196X; Santa Cruz Biotechnology), aP2 (goat sc-18661; Santa Cruz Biotechnology), C/EBPα(rabbit sc-61X; Santa Cruz Biotechnology), β-actin (A5316; Sigma-Aldrich), MG132 (M-1157, A.G.Scientific, San Diego, CA, USA), and geldanamycin (9843 S, Cell Signaling Technology). Pepstatin A (P5318), aprotinin (A1153), phenylmethanesulfonylfluoride (PMSF; P7626), leupeptin (L2884), dimethyl sulfoxide (DMSO; D8418), N-ethylmaleimide (NEM; E3876), dexamethasone (D1756), 3-isobutyl-1-methylxanthine (IBMX; I5879), and Oil Red O (O0625) were purchased from Sigma-Aldrich (St. Louis, MO, USA), while insulin (11 376 497 001) was purchased from Roche (Mannheim, Germany).

Mouse anti-ZO-1 monoclonal antibody (mAb) (T8/754), rat anti-occludin mAb (MOC37), and rabbit anti-claudin-2 polyclonal antibody (pAb) were characterized as described previously [41 (link)–43 (link)]. Mouse anti-claudin-2 mAb (32–5600), rabbit anti-claudin-3 pAb (34–1700), alexa fluor 488 phalloidin (A12379) and alexa fluor 647 phalloidin (A22287) were purchased from Life Technologies. Mouse anti-E-cadherin mAb (ECCD-2; M108) was purchased from Clontech. Mouse anti-Na⁺/K⁺ ATPase α1 mAb (NB300-146) was purchased from Novus Biologicals. Fluorescein isothiocyanate-dextran (FITC-dextran), benzamil (B2417), bumetanide (B3023), ouabain (O3125), H89 (B1427), genistein (G6649), Go6983 (G1918) and 3-isobutyl-1-methylxanthine (IBMX, I5879) were purchased from Sigma Aldrich. Fluorescein (16106–82) was purchased from Nacalai tesque. SQ22536 was purchased from Santa Cruz. Forskolin was purchased from Wako.

The 3T3‐L1 pre‐adipocytes were seeded onto a 6‐well plate at 10⁵ cells/well. Complete culture medium was added, and the cells were cultured at 37℃ with 5% CO₂. The medium was changed every 2 days. After 2 days, complete culture containing 0.5 mM 3‐Isobutyl‐1‐methylxanthine (IBMX, I5879; Sigma‐Aldrich, St. Louis, MO, USA ), 1 µM dexamethasone (D4902; Sigma‐Aldrich) and 5 µg/mL insulin (HI0240; Eli Lilly, Indianapolis, IN, USA) were added (2 mL/well), and the cells were incubated for 48 h. The medium was replaced with a complete culture medium containing 5 µg/mL insulin (2 mL/per well). After 48 h, culture was continued using the complete culture medium, and the medium was changed every 2 days. At 8‐10 days after induced differentiation, 80%‐90% of 3T3‐L1 pre‐adipocytes showed an adipocyte phenotype. Morphology was examined over 10 days using oil red O staining and light microscopy (Nikon, Tokyo, Japan).