Journal: Journal of Autoimmune Diseases

Article Title: Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis

doi: 10.1186/1740-2557-1-4

Figure Lengend Snippet: Immunoblot patterns produced by anti-α-enolases and anti-tRNP (Ser)Sec antibodies on electrophoretically separated primate and rat liver homogenates and dot blot results with recombinant tRNP (Ser)Sec . In both rat and primate liver preparations, a band of ~48 kDa is immunofixed by a polyclonal goat IgG anti-α-enolase specific antibody; a band of ~50 kDa is immunofixed by a serum containing a high-titre anti-tRNP (Ser)Sec antibody. Anti-α-enolase antibody does not recognize tRNP (Ser)Sec by dot blot analysis.

Article Snippet: An affinity purified goat polyclonal anti-α-enolase IgG antibody raised against a peptide mapping near the carboxyl-terminus of human α-enolase, which is common to α, β, and γ isoforms of mouse, rat and human enolase (200 μg/ml; Santa Cruz Biotechnology, Santa Cruz, California, USA) was used as reference serum sample at a dilution of 1:100, according to the manufacturer's instructions.

Techniques: Western Blot, Produced, Dot Blot, Recombinant

Journal: Journal of Autoimmune Diseases

Article Title: Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis

doi: 10.1186/1740-2557-1-4

Figure Lengend Snippet: Immunoblot patterns produced on rat liver homogenate by (lane 1) a polyclonal goat IgG anti-α-enolase specific antibody; (lanes 2–5) four representative anti-soluble liver antigen (SLA) positive sera; (lane 6) a reference serum containing high-titre anti-tRNP (Ser)Sec antibody.

Article Snippet: An affinity purified goat polyclonal anti-α-enolase IgG antibody raised against a peptide mapping near the carboxyl-terminus of human α-enolase, which is common to α, β, and γ isoforms of mouse, rat and human enolase (200 μg/ml; Santa Cruz Biotechnology, Santa Cruz, California, USA) was used as reference serum sample at a dilution of 1:100, according to the manufacturer's instructions.

Techniques: Western Blot, Produced

Journal: Journal of Autoimmune Diseases

Article Title: Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis

doi: 10.1186/1740-2557-1-4

Figure Lengend Snippet: Immunoblot patterns obtained using 9 SLA positive and 9 SLA negative serum samples against recombinant α-enolase. A polyclonal goat IgG anti-α-enolase specific antibody has been used as a reference positive serum. Ab, antibody; ag, antigen

Article Snippet: An affinity purified goat polyclonal anti-α-enolase IgG antibody raised against a peptide mapping near the carboxyl-terminus of human α-enolase, which is common to α, β, and γ isoforms of mouse, rat and human enolase (200 μg/ml; Santa Cruz Biotechnology, Santa Cruz, California, USA) was used as reference serum sample at a dilution of 1:100, according to the manufacturer's instructions.

Techniques: Western Blot, Recombinant

Journal: Breast Cancer Research

Article Title: Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells

doi: 10.1186/bcr1392

Figure Lengend Snippet: Lack of acinar overgrowth in MCF-10A cells expressing kinase-dead IGFIR. (a) Differential interference contrast images of acinar structures formed from stable pools of MCF-10A cells infected with virus expressing empty vector (pCLXSN; panels a, b), kinase-dead (K1003A; panels d, e) and wild-type IGFIR (panels g, h); scale bar = 80 μm. Equatorial confocal sections of pCLXSN (panel c), K1003A (panel f) and wild-type IGFIR (panel i) acinar structures labeled with antibodies to the IGFIR (green) and the nuclear counterstain TO-PRO-3 (blue); scale bar = 40 μm. Cells were grown for 20 days in media supplemented with 10 μg/ml insulin (panels a, c, d, f, g, i) or 100 ng/ml IGF-I (panels b, e, h). (b) Western blots of whole-cell lysates from monolayer cultures of MCF-10A cells expressing empty vector (pCLXSN), kinase-dead (K1003A) or wild-type IGFIR (IGFIR) untreated or treated with 50 ng/ml IGF-I (left panel) or 10 μg/ml insulin (right panel) for 5 minutes, 60 minutes or overnight (o/n) after overnight incubation in serum-free media. Proteins were separated by 12.5% SDS-PAGE, transferred to PVDF and incubated overnight with the indicated antibodies to signaling proteins downstream of IGFIR activation.

Article Snippet: Antibodies to the carboxyl terminus of the β-subunit of the IGFIR were described previously [ ]; extracellular-signal regulated kinase (ERK)2, p70S6 kinase (p70S6K1) and IRS-1 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); phospho-Akt (Ser473), phospho-Akt (Ser473 Immunohistochemistry specific), Akt, phospho-mammalian target of rapamycin (mTOR; Ser2448) and cleaved caspase-3 (Asp175) were from Cell Signaling (Beverly, MA, USA); phospho-ERK1/2 and α-tubulin were from Sigma; laminin V (γ2 chain) was from Chemicon International (Temecula, CA, USA); E-cadherin and Golgi matrix protein of 130 kDa (GM130) were from BD Transduction Laboratories (Franklin Lakes, NJ, USA; Ki-67 was from Zymed Laboratories (San Francisco, CA, USA); and phospho-p70S6K1 (Thr 389 ) was from Upstate (Charlottesville, VA, USA).

Techniques: Expressing, Infection, Plasmid Preparation, Labeling, Western Blot, Incubation, SDS Page, Activation Assay

Journal: Breast Cancer Research

Article Title: Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells

doi: 10.1186/bcr1392

Figure Lengend Snippet: Increased proliferation and decreased luminal apoptosis in IGFIR structures. (a) Confocal sections of 34-day acinar structures from control (pCLXSN; panels a, c), and IGFIR-expressing (panels b, d) MCF-10A cells labeled with antibodies to the proliferative marker Ki-67 (green; panels a, b) or cleaved caspase-3 (green; panels c, d) and the nuclear counterstain TO-PRO-3 (blue). Scale bars = 50 μm. Arrows indicate a Ki-67-labeled cell (panel a) and a cleaved caspase-3 labeled cell (panel d). (b) Bar graph showing the percent of acini with three or more Ki-67-positive cells in control and IGFIR-expressing structures. Data are mean ± standard deviation from three different experiments grown for 20 days from two independently derived cell lines. At least 100 acini were counted in each experiment; the asterisk indicates significance, p < 0.01, Student's t test.

Article Snippet: Antibodies to the carboxyl terminus of the β-subunit of the IGFIR were described previously [ ]; extracellular-signal regulated kinase (ERK)2, p70S6 kinase (p70S6K1) and IRS-1 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); phospho-Akt (Ser473), phospho-Akt (Ser473 Immunohistochemistry specific), Akt, phospho-mammalian target of rapamycin (mTOR; Ser2448) and cleaved caspase-3 (Asp175) were from Cell Signaling (Beverly, MA, USA); phospho-ERK1/2 and α-tubulin were from Sigma; laminin V (γ2 chain) was from Chemicon International (Temecula, CA, USA); E-cadherin and Golgi matrix protein of 130 kDa (GM130) were from BD Transduction Laboratories (Franklin Lakes, NJ, USA; Ki-67 was from Zymed Laboratories (San Francisco, CA, USA); and phospho-p70S6K1 (Thr 389 ) was from Upstate (Charlottesville, VA, USA).

Techniques: Expressing, Labeling, Marker, Standard Deviation, Derivative Assay

Journal: Breast Cancer Research

Article Title: Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells

doi: 10.1186/bcr1392

Figure Lengend Snippet: Disrupted apico-basal polarity of IGFIR structures. Equatorial confocal sections of 20-day control (pCLXSN, panels a, c, e) and IGFIR structures (panels b, d, f) labeled with antibodies to laminin V (red; panels a, b), E-cadherin (red; panels c, d) and Golgi matrix protein of 130 kDa (GM130; green; panels e, f) and the nuclear counterstain TO-PRO-3 (blue). Arrows indicate localization of E-cadherin at cell-cell junctions (panel d) and apical localization of GM130 in control structures (panel e). Arrowheads show sites of punctate E-cadherin labeling (panel d). Scale bars = 50 μm.

Article Snippet: Antibodies to the carboxyl terminus of the β-subunit of the IGFIR were described previously [ ]; extracellular-signal regulated kinase (ERK)2, p70S6 kinase (p70S6K1) and IRS-1 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); phospho-Akt (Ser473), phospho-Akt (Ser473 Immunohistochemistry specific), Akt, phospho-mammalian target of rapamycin (mTOR; Ser2448) and cleaved caspase-3 (Asp175) were from Cell Signaling (Beverly, MA, USA); phospho-ERK1/2 and α-tubulin were from Sigma; laminin V (γ2 chain) was from Chemicon International (Temecula, CA, USA); E-cadherin and Golgi matrix protein of 130 kDa (GM130) were from BD Transduction Laboratories (Franklin Lakes, NJ, USA; Ki-67 was from Zymed Laboratories (San Francisco, CA, USA); and phospho-p70S6K1 (Thr 389 ) was from Upstate (Charlottesville, VA, USA).

Techniques: Labeling

Journal: Breast Cancer Research

Article Title: Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells

doi: 10.1186/bcr1392

Figure Lengend Snippet: Induction of Akt signaling in IGFIR structures. Equatorial confocal sections of 34-day control (pCLXSN, panels a, c) and IGFIR acinar structures (panels c, d) labeled with phosphospecific antibodies to Akt- [S473] (green; panels a, b) and mammalian target of rapamycin (mTOR)- [S2448] (green; panels c, d) and the nuclear counterstain TO-PRO-3 (blue). Scale bars = 50 μm.

Article Snippet: Antibodies to the carboxyl terminus of the β-subunit of the IGFIR were described previously [ ]; extracellular-signal regulated kinase (ERK)2, p70S6 kinase (p70S6K1) and IRS-1 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); phospho-Akt (Ser473), phospho-Akt (Ser473 Immunohistochemistry specific), Akt, phospho-mammalian target of rapamycin (mTOR; Ser2448) and cleaved caspase-3 (Asp175) were from Cell Signaling (Beverly, MA, USA); phospho-ERK1/2 and α-tubulin were from Sigma; laminin V (γ2 chain) was from Chemicon International (Temecula, CA, USA); E-cadherin and Golgi matrix protein of 130 kDa (GM130) were from BD Transduction Laboratories (Franklin Lakes, NJ, USA; Ki-67 was from Zymed Laboratories (San Francisco, CA, USA); and phospho-p70S6K1 (Thr 389 ) was from Upstate (Charlottesville, VA, USA).

Techniques: Labeling

Journal: Breast Cancer Research

Article Title: Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells

doi: 10.1186/bcr1392

Figure Lengend Snippet: Inhibition of PI3K or MEK but not mTOR disrupts IGFIR acinar development. (a) Confocal sections of 20-day control (pCLXSN, panels a, d, g) and IGFIR (panels b, c, e, f, h, i) acinar structures grown in the presence of the MEK inhibitor, UO126 (10 μm; panels a-c), the PI3K inhibitor LY294002 (50 μm; panels d-f) and the mTOR inhibitor rapamycin (40 nM; panels g-i). Fixed structures were labeled with the nuclear counterstain TO-PRO-3 (blue) and with antibodies to Ki-67 (green; panels a, b, d, e, g, h) or cleaved caspase-3 (green; panels c, f, i). Scale bars = 50 μm. The arrow in panel b indicates Ki-67-labeled cells. (b) Whole-cell lysates of 16d IGFIR acinar structures were isolated after overnight serum starvation followed by 30 minute pretreatment with DMSO (vehicle) or 40 nM rapamycin and subsequent 30-minute incubation with 10 μg/ml insulin. Lysates were immunoprecipitated with antibodies to p70S6K, separated by 12.5% SDS-PAGE, transferred to PVDF and immunoblotted with antibodies to p70S6K or phosphospecific antibodies to Thr389 of p70S6K as indicated.

Article Snippet: Antibodies to the carboxyl terminus of the β-subunit of the IGFIR were described previously [ ]; extracellular-signal regulated kinase (ERK)2, p70S6 kinase (p70S6K1) and IRS-1 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); phospho-Akt (Ser473), phospho-Akt (Ser473 Immunohistochemistry specific), Akt, phospho-mammalian target of rapamycin (mTOR; Ser2448) and cleaved caspase-3 (Asp175) were from Cell Signaling (Beverly, MA, USA); phospho-ERK1/2 and α-tubulin were from Sigma; laminin V (γ2 chain) was from Chemicon International (Temecula, CA, USA); E-cadherin and Golgi matrix protein of 130 kDa (GM130) were from BD Transduction Laboratories (Franklin Lakes, NJ, USA; Ki-67 was from Zymed Laboratories (San Francisco, CA, USA); and phospho-p70S6K1 (Thr 389 ) was from Upstate (Charlottesville, VA, USA).

Techniques: Inhibition, Labeling, Isolation, Incubation, Immunoprecipitation, SDS Page