biotinylated anti igf ii antibody  (R&D Systems)

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: G3: Genes|Genomes|Genetics

Article Title: Impact of Genetic Background on Neonatal Lethality of Gga2 Gene-Trap Mice

doi: 10.1534/g3.114.010355

Figure Lengend Snippet: Analysis of serum IGF-II and brain IGF-II receptor (CI-MPR) levels. (A) Serum IGF-II levels of newborn mice were determined by ELISA as described in Materials and Methods . The serum obtained from each pup was sufficient to perform the assay in duplicate. Results are mean ± SD, n = 16 per group. (B) Western blot analysis of brain tissue from two wt and two Tigm Gga2 −/− mice in the mixed background. Twenty-five μg protein extract for each sample was subjected to SDS-PAGE and immunoblot analysis of CI-MPR, SorLA, and Sortilin. GAPDH (5 μg of lysate) was included as a loading control. NS, not significant as determined by Student t test.

Article Snippet: Standard, control, or samples (100 μl/well) were combined with 50 ng (100 μl) biotinylated goat anti-mouse IGF-II antibody (R&D) and incubated for 3 hr on a shaker at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, SDS Page, Control