Anti creb 48h2

Fetal bovine serum (FBS) was purchased from Cytiva (Marlborough, MA, USA). Reverse transcriptase and SYBR green reagents were obtained from ThermoFisher (ThermoFisher Scientific, Waltham, MA, USA). Antibodies were obtained from different sources: anti-β-actin (Novus Cat. # NB600-501; St. Louis, MO, USA), anti-FGF21 (abcam Cat. #ab171941), anti-phospho-Akt (Ser473) (Cell Signaling, Cat. # 9271; Danvers, MA, USA), anti-Akt (Cell Signaling, Cat. # 4691), anti-phospho-CREB (87G3) (Cell Signaling, Cat. # 9197), anti-CREB (48H2) (Cell Signaling Cat. # 9197), anti-phospho-JNK (Thr183/Thr185) (Cell Signaling, Cat. # 9251), anti-JNK (Cell Signaling, Cat. # 9258), anti-phospho-p38 (Thr182) (Santa Cruz Cat. # sc-7973), anti-p38 (Cell Signaling, Cat. # 9212), anti-phospho-NF-κB p65 (S536) (R&D Systems, Cat. # KCB7226; Minneapolis, MN, USA), and anti-NF-κB p65 (Cell Signaling, Cat. # 8242). The materials for the animal diets were obtained from Research Diets Inc. (New Brunswick, NJ, USA). Glucosamine hydrochloride (Cat. 66842), along with all other unspecified chemicals and reagents utilized in this project, was procured from Sigma-Aldrich (St. Louis, MO, USA).

MCF-7 cells were washed twice with PBS and then mechanically detached with PBS. One third of the cells were centrifuged and solubilized in RIPA buffer for the total fraction. The remaining two thirds were centrifuged at 1200 rpm for 10 min at 4°C. Pellets for fractionation were resuspended in hypotonic buffer (10 mM Tris, pH 8.0, 10 mM KCl, 2 mM phenylmethylsulfonyl fluoride plus protease inhibitor mixture) to allow cell swelling for 2 min at 4°C. Then, Nonidet P-40 was added to a final concentration of 0.4%. Samples were centrifuged at 2,000 rpm for 5 min at 4°C and the supernatants collected as the cytoplasmic fractions. Pellets containing cell nuclei were washed once with hypotonic buffer and then extracted with high salt lysis buffer (50 mM Tris pH 8.0, 5 mM EDTA, pH 8.0, 1% Nonidet P-40, 400 mM NaCl, 2 mM phenylmethylsulfonyl fluoride plus protease inhibitor mixture) and sonicated. Equal amounts of proteins were loaded onto SDS acrylamide gel, transferred onto nitrocellose membranes, and blotted with anti-DDR1 (C-20), anti IGF-IR (Cell Signaling Technology), anti-β-tubulin, and anti-CREB 48H2 (Cell Signaling Technology) antibodies.

Western blot analysis was performed as described previously.^{15 (link)} For detection of phosphoproteins, PhosStop (Roche, Penzberg, Germany) was added to the lysis buffer. The following antibodies were used for detection: anti-HDAC8 (H-145) (polyclonal; Santa Cruz, Santa Cruz, CA, USA), anti-acetyl tubulin (clone 6-11B-1; Sigma), anti-acetyl-histone H4 (polyclonal; Upstate, Lake Placid, NY, USA), anti-acetyl-SMC3 (provided by Prof. K Shirahige, University of Tokyo, Tokyo, Japan), anti p21^waf1/cip1 (clone CP74; Merck), anti-Trk (C-14) (polyclonal; Santa Cruz), anti-neurofilament-M (polyclonal; Merck), anti-β-actin (clone AC-15; Sigma), anti-P-CREB (Ser133; Cell Signaling, Danvers, MA, USA), anti-CREB (48H2; Cell Signaling) and anti-GAPDH (clone 6C5; Merck).