Journal: Hepatology (Baltimore, Md.)

Article Title: Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy.

doi: 10.1002/hep.26996

Figure Lengend Snippet: Fig. 2. Isolation of a human antibody targeting HS chains of GPC3. (A) The binding affinity of HS20 on GPC3-his. (B) The binding affinity of HS20 on A431-GPC3 cells. (C) Flow cytometry analysis of HS20 on A431 and A431-GPC3 cells. (D) Binding properties of HS20 on GPC3, GPC3DHS, or irrelevant human Fc protein IAB. 1G12, a commercial mouse antibody against GPC3. (E) Immunoprecipitation of GPC3 in A431- GPC3 cells, Hep3B, and Hep3B GPC3 knockdown cells. Cell lysates incubated with HS20 or human IgG for immunoprecipitation. The immuno- precipitated complex was detected by 1G12. Black arrow indicates the GPC3 core protein and the bracket indicates glycosylated GPC3. Wt: wild-type; ko: knockdown. GPC3 knockdown efficiency is shown in Fig. 5C. (F) Competitive ELISA. Different concentrations of GPC3 or GPC3DHS were preincubated with HS20 (0.1 nM) for 1 hour. The mixture was added into the GPC3-coated plate to measure the binding of HS20/GPC3. CD22 was used as the negative control. The data represent the mean 6 SD.

Article Snippet: HepG2, Hep3B, and A431 (human epithelial carcinoma) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Isolation, Binding Assay, Flow Cytometry, Immunoprecipitation, Knockdown, Incubation, Competitive ELISA, Negative Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy.

doi: 10.1002/hep.26996

Figure Lengend Snippet: Fig. 3. Both sulfated and nonsulfated regions of HS chain are involved in HS20 binding. (A) Treatment of GPC3-expressing cells with heparinase I, II, and III. A431-GPC3 cells were treated with different heparinases for 2 hours at 37C. Cells were then stained with HS20 (5 lg/mL) or YP7 (1 lg/mL) for flow cytometry. (B) Statistical analy- sis of three independent experiments. The binding fluorescence signals of untreated cells represent 100%. The data represent the mean 6 SD of three independent experiments (**P < 0.01 and #P > 0.05).

Article Snippet: HepG2, Hep3B, and A431 (human epithelial carcinoma) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Binding Assay, Expressing, Staining, Cytometry

Journal: BMC cancer

Article Title: Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer.

doi: 10.1186/1471-2407-13-518

Figure Lengend Snippet: Figure 5 EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signal- ling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).

Article Snippet: Whole cell lysate of EGF-treated A431 epithelial carcinoma cells used as positive control was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Positive Control, Control, Software

Journal: Bioconjugate chemistry

Article Title: Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

doi: 10.1021/acs.bioconjchem.5b00613

Figure Lengend Snippet: Fig. 6. Detection of proteins in cell lysates. A panel of 24-plex antibody binders was used to detect their respective target proteins in MCF7, K562, A549, and A431 cell lysates. The cell lysates were diluted to different concentrations and reacted with a mixture of antibody binders. Only selected examples of protein targets CSTB (A), CASP3 (B), Ki-67(C), and GATA3 (D) are shown. Ct values were shown as the means ± SD. Ct: cycle threshold.

Article Snippet: MCF7, K562, A549, and A431 cell lysates were obtained from Novus (Littleton, CO).

Techniques:

Journal: The Journal of biological chemistry

Article Title: Modulation of epidermal growth factor receptor gene transcription by a polymorphic dinucleotide repeat in intron 1.

doi: 10.1074/jbc.274.19.13176

Figure Lengend Snippet: FIG. 3. In vitro run-off transcription of the EGFR gene. A, EGFR 4,050-bp PCR products from the indicated cell lines were used as templates for run-off transcription with A431 nuclear extracts. After transcription, DNA was digested with PstI, electrophoresed through a denaturing 4% polyacrylamide, 13 TBE gel, and transferred to a nylon membrane. RNA products and DNA fragments were detected after hybridization with a fluoresceinated RNA probe. The DNA template is detected in fragments of 1,051, 848, 706, and 412 nt, and run-off transcripts are sized approximately 1,650 and 1,550 nt. The sizes of an RNA standard are shown on the left side. Controls without DNA tem- plate and with RNase treatment in the right lanes revealed no RNA products. B, determination of the ratio of RNA product intensity to the DNA 1,051-bp band intensity dependent on the DNA template amount. The ratio is approximately constant between 10 and 100 ng DNA.

Article Snippet: Approximately 50 ng of PCR product was combined with 6 ml of 13 transcription buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 0.5 mM dithiothreitol, 0.2 mM EDTA, 20% glycerol, in diethyl pyrocarbonate-treated water), 1 ml of RNasin, 2 ml of 50 mM MgCl2, 1 ml of 10 mM NTP mix (all Promega), 5 ml of A431 nuclear extract (25 mg of protein, Santa Cruz Biotechnology), and diethyl pyrocarbonate-treated water to a final volume of 25 ml.

Techniques: In Vitro, Membrane, Hybridization

Journal: Hepatology (Baltimore, Md.)

Article Title: Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats.

doi: 10.1053/jhep.2002.31355

Figure Lengend Snippet: Fig. 3. Group phenotyping of LPC strains and clonal lines cultured on STO feeder layers. Cellular extracts from 22 sources were made 3 days postplating and analyzed (lanes 4-25) for AFP, albumin, CK14, c-kit, CD34, and desmin by Western blotting. The names of individual cell strains and cell lines are shown above each lane; the larger single numbers above the names designate group assignments (see Table 2). Extracts used as internal controls were obtained from neonatal liver (BRL) and, as indicated, from bone marrow and skeletal muscle (lane 1), STO fibroblasts (lane 2), freshly isolated adult rat hepatocytes and, as annotated, human A431 cells (lane 3). The 6 positive control markers included AFP, albumin, CK14, and c-kit (BRL), CD34 (bone marrow), and desmin (skeletal muscle) in lane 1; and CK14 (A431 cells) and albumin (adult hepatocytes) in lane 3. Negative controls were provided by STO cells for all markers (lane 2), by adult rat hepatocytes for AFP and c-kit (lane 3), and for CK14 (not shown). Molecular weights of the 6 markers are listed to the right of lane 25.

Article Snippet: The following materials were obtained commercially: dexamethasone and bFGF (Sigma); Oncostatin M (R&D Systems, Minneapolis, MN); Matrigel (Collaborative Research, Bedford, MA); human epidermoid carcinoma cell line A431 lysates (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Cell Culture, Western Blot, Isolation, Positive Control