Journal: Journal of Cancer Prevention

Article Title: Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells

doi: 10.15430/JCP.2014.19.3.161

Figure Lengend Snippet: Effect of kaempferol on the expression of insulin-like growth factor (IGF)-II and heregulin (HRG) and the phosphorylation of Akt and extracellular regulated kinase (ERK)- 1/2. HT-29 cells were serum-starved and treated with various concentrations (0, 20, 40, or 60 μmol/L) of kaempferol. (A) At 24 hours after treatment, 24 hour-conditioned media were collected and subjected to Western blot analysis with anti-IGF-II antibody. Media loaded onto the gel was adjusted for equivalent cell numbers. (B) At the indicated time point after kaempferol treatment, total cell lysates were prepared and subjected to Western blot analysis with their relevant antibodies. (C, D) At 2 hours after kaempferol treatment, total cell ly-sates were prepared. (C) Total cell lysates were subjected to Western blot analysis with the relevant antibodies. (D) Total cell lysates were incubated with immobilized P-ERK- 1/2 antibody overnight (4 ° C). After centrifugation, the immunoprecipitated proteins were incubated with Elk-1 (an ERK-1/2 substrate) and ATP for 30 minutes (30 ° C). The resulting P-Elk-1 was analyzed by Western blot analysis with an anti-P-Elk antibody. IP, immunoprecipitation; WB, western blot analysis.

Article Snippet: Kaempferol, essentially fatty acid-free bovine serum albumin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 7-amino-actinomycin D (7-AAD), phosphatidylinositol, transferrin and anti-β-actin antibody were obtained from Sigma (St. Louis, MO, USA); selenium and DMEM/Ham’s F-12 nutrient mixture (DMEM/F-12 were purchased from Gibco BRL (Gaithersburg, MD, USA)); [methyl- 3 H]thymidine, protein A-Sepharose and horse-radish peroxidase-conjugated anti-rabbit and anti-mouse IgG were from Amersham (Arlington Heights, IL, USA); antibodies against HRG, IGF-IRβ, p85 and ErbB3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Akt, phospho (P)-Akt, ERK-1/2 and P-ERK-1/2 were from Cell Signaling Technology (Beverly, MA, USA); anti-phosphotyrosine-RC20 (PY20) antibody linked to horse-radish peroxidase was purchased from BD Transduction Laboratories (Palo Alto, CA, USA); anti-IGF-II antibody was from Amano International Enzyme (Troy, VA, USA); recombinant human HRG-β and recombinant human IGF-II were purchased from R&D Systems, (Minneapolis, MN, USA); phycoerythrin-conjugated Annexin V was from BD Pharmingen, (Franklin Lake, NJ, USA); and [ γ - 32 P]ATP was obtained from PerkinElmer Life and Analytical Sciences, Inc. (Boston, MA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Incubation, Centrifugation, Immunoprecipitation

Journal:

Article Title: IGF-2 and FLT-1/VEGF-R1 mRNA levels reveal distinctions and similarities between congenital and common infantile hemangioma

doi: 10.1203/PDR.0b013e318163a243

Figure Lengend Snippet: IGF-2 protein localized in relation to blood vessels in common and congenital hemangioma. A–C) CD31 immunostaining of frozen sections highlights blood vessels in proliferating common hemangioma (A), NICH (B) and RICH (C). Nuclei are stained blue with DAPI. D–E) Common hemangioma sections double-labeled with anti-IGF2 (green) and anti-CD31 (red). Nuclei stained with DAPI (blue). Panel D, a proliferating phase hemangioma while Panel E, involuting phase hemangioma. F) RICH double-labeled with anti-IGF2 (green) and anti-CD31 (red). G) NICH stained with anti-IGF2 (green). H) Pyogenic granuloma stained with anti-IGF2 (green) as negative control. Scale bars are 50 µm. Panel A is 200X magnification; Panels B, C, G and H, 100X magnification; Panels D–F, 300X magnification.

Article Snippet: Indirect immunofluorescence Cryosections ( 4 μm ) of the tumors (proliferating and involuting infantile hemangioma), RICH, NICH and pyogenic granulomas were fixed with cold acetone for 15 min. After washing for 5 min in PBS, and incubating with species appropriate blocking serum, the following primary antibodies were used: mouse anti-human IGF2 mAb 1:50 (R&D Systems, USA), mouse anti-human CD31 mAb 1:50 (DAKO, Carpinteria, CA, USA), goat anti-human CD31 antibody 1:50 (Santa Cruz Biotechnology Inc., CA, USA).

Techniques: Immunostaining, Staining, Labeling, Negative Control