Anti cyclin d1

Murrayafoline A [Brown oil, C₁₄H₁₃NO, Rf: 0.25 (hexane/EtOAc, 10: 0.5), EI-MS m/z: 211 (100%) 196 (M-CH₃)⁺, 167, 139, 115, 101, 77] was obtained as previously described [17 (link)]. The structure of murrayafoline A was established by ¹H- and ¹³C-NMR analysis. The purity of murrayafoline A was estimated to be higher than 97% by both HPLC and spectroscopic analysis. All cell culture materials were purchased from Invitrogen (Carlsbad, CA, USA). Anti-phospho-ERK1/2, anti-phospho-PLCγ1, anti-phospho-PDGF-R β (Tyr⁷⁵¹), anti-phospho-STAT3 (Tyr⁷⁰⁵), anti-ERK1/2, anti-Akt, anti-PLCγ1, and anti-PDGF-Rβ antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-phospho-Akt antibodies were purchased from Millipore Corp. (Billerica, MA, USA). Anti-phospho-pRb, anti-CDK2, anti-CDK4, anti-phospho PCNA, anti-cyclin D1, anti-cyclin E, anti-Akt, and anti-β-actin antibodies were purchased from Abfrontier (Geumcheon, Seoul, Korea). PDGF-BB was obtained from Upstate Biotechnology (Lake Placid, NY, USA). All other chemicals used were of analytical grade.

Rat liver tissues and cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma-Aldrich). A total of 45 μg protein was separated in sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). The separated proteins were transferred onto PVDF membranes (Bio-Rad). The membranes were incubated with anti-CRP (1：1,000, Abcam Inc.), anti-VEGF (R&D system), anti-VEGFR2 (R&D system), anti-β-catenin (active form, Cell Signaling), anti-pGSK3 (Cell Signaling), anti-β-catenin (active form, Cell Signaling), anti-albumin (Novus), anti-HNF1α (Abcam Inc.), anti-cyclinD1 (AbFrontier), anti-actin (Sigma-Aldrich), anti-tubulin (Abcam Inc.), and anti-GAPDH (AbFrontier) antibodies at 4℃ over-night. The membranes were then incubated with horseradish peroxidase-conjugated secondary anti-mouse IgG (Cell Signaling), anti-rabbit IgG (Cell Signaling) or anti-goat IgG (Santa Cruz) antibody for 1 hour at RT. Bands were detected using Clarity Western ECL kit (Bio-Rad).