Anti mouse igg w402b

RPMI1640/L-glutamine, DMEM/High glucose, 2β-mercaptoethanol (35602), antibiotic-antimycotic (SV30079.01) and Supersignal West Pico Chemiluminescence Substrate (34080) were purchased from Thermo Scientific. Sodium pyruvate (25-000-CI) was purchased from Mediatech Inc. Fetal Bovine Serum (FBS) (S01520) was purchased from Biowest. Metafectene transfection reagent (T020) was purchased from Biontex Laboratories (Germany). ³[H]-2-deoxyglucose was purchased from MP Biochemicals. TGF-β1 (240-B), TGF-β3 (243-B3), Nodal (1315-ND-025CF) and Activin A (338-AC) were obtained from R&D Systems. TGF-β2 was kindly provided by Dr. Steven Ledbetter (Genzyme). SB 431542 (#1614) was purchased from Tocris. The 3TP-lux reporter has been previously described (22 (link), 23 (link)). Anti-Smad2 rabbit polyclonal (51-1300), rabbit polyclonal anti-Smad3 (51-1500), were purchased from Zymed. Rabbit polyclonal anti-phosphoSmad3 was a kind gift of Dr. Ed Leof, Mayo Clinic (24 (link)). Mouse monoclonal anti-Smad4 (B8)(sc-7966) and rabbit polyclonal anti-phosphoSmad2 (06-829) were purchased from Santa Cruz Biotechnology and Upstate Biotechnology, respectively. The secondary antibodies anti-rabbit IgG (W401B) and anti-mouse IgG (W402B) were purchased from Promega. The pcDNA3 vector was obtained from Invitrogen.

Real-time PCR analysis was performed with primers and probe sets from Applied Biosystems, as described (Liu et al., 2009 (link)). Immunoblots were performed as described previously (Liu et al., 2010 (link)), using the primary antibodies anti-CD115, anti-phosphorylated (T37/46) 4E-BP1 (236B4), anti-phosphorylated (S235/236) S6 (DX57.2.2E), anti-phosphorylated ERK1/2 (D13.14.4E), anti-phosphorylated (S473) AKT (D9E), anti-S6 (5G10), anti-4E-BP-1 (53H11), anti-ERK1/2 (137F5), anti Akt (polyclonal; all from Cell Signaling Technology), anti-SREBP2 (ab30682; Abcam), and anti-β-actin (AC-74; Sigma-Aldrich) and the secondary horseradish peroxidase–conjugated antibodies anti–rabbit IgG (W401B) and anti–mouse IgG (W402B; both from Promega).

Standard Western blotting procedure was used. Primary antibodies: NPY1R (Abcam, NPY1R11-A), NPY5R (Abcam, NPY5R11-A), HIF-1α (Novus Biological, NB100-123), HIF-2α (Novus Biological, NB100-122), Phospho-p44/42 MAPK (T202/Y204) (Cell Signaling, 4370S), p44/42 MAPK (Erk1/2) (Cell Signaling, 4695S), GAPDH (Cell Signalling, 5174S). HRP-conjugated antibodies: Promega anti-Rabbit IgG (W401B) and anti-mouse IgG (W402B). Densitometry was performed using Image Lab (Bio-Rad).