Journal:

Article Title: Prolactin Inhibits Activity of Pyruvate Kinase M2 to Stimulate Cell Proliferation

doi: 10.1210/me.2010-0219

Figure Lengend Snippet: A, Whole-cell lysates from human embryonic kidney 293T cells transfected to express Flag-tagged PRLr or the corresponding control vector (pCDNA3) and treated with PRL were immunoprecipitated (IP) with anti-Flag M2 agarose followed by stringent washes to minimize nonspecific interactions. The proteins that copurified with PRLr were resolved by SDS-PAGE and visualized by Colloidal Coomassie staining. Indicated proteins (Band 1) were excised, digested with trypsin, and analyzed by LC-MS/MS. The results were searched against the NIH database using SEQUEST software. B, Material from the experiment shown in panel A was analyzed by immunoblotting (IB) using anti-Flag and anti-PKM2 antibodies. C, Lysates from the MCF10a-Δp53 cells stably expressing Flag-PRLr [wild type (WT) or SA mutant], previously characterized in Ref. 12 were immunoprecipitated using Flag antibody and analyzed by IB using anti-PRLr antibody or anti-PKM2 antibody. Levels of PKM2 in the whole-cell extracts (WCE) are also shown. D, 293T cells transfected with Flag-tagged PRLr and HA-tagged PKM2 as indicated were lysed and IP-IB assays using anti-Flag and anti-HA antibody were carried out as depicted.

Article Snippet: Conversely, PRLr immunoprecipitated with N30 antibody was detected by commercially available monoclonal anti-PRLr antibody (Fig. 2B ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 A, Immunoprecipitation (IP) of endogenous PRLr from lysates from 293T cells treated with or without human PRL (purchased from the National Hormone and Peptide program and used at 100 ng/ml for 30 min) was carried out using anti-PRLr antibody (H-300, Santa Cruz) or naïve rabbit serum (NRS).

Techniques: Transfection, Control, Plasmid Preparation, Immunoprecipitation, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Software, Western Blot, Stable Transfection, Expressing, Mutagenesis

Journal:

Article Title: Prolactin Inhibits Activity of Pyruvate Kinase M2 to Stimulate Cell Proliferation

doi: 10.1210/me.2010-0219

Figure Lengend Snippet: A, Immunoprecipitation (IP) of endogenous PRLr from lysates from 293T cells treated with or without human PRL (purchased from the National Hormone and Peptide program and used at 100 ng/ml for 30 min) was carried out using anti-PRLr antibody (H-300, Santa Cruz) or naïve rabbit serum (NRS). Levels of PKM2 in whole-cell extracts (WCE) are also shown. B, MDA-MB-231 cells were infected with adenoviruses for delivery of human PRLr at increasing multiplicity of infection (MOI). WCEs were prepared and aliquots were resolved by SDS-PAGE and subjected to direct immunoblotting (IB) using anti-PRLr (N30, upper panel). Additional aliquots of the extracts were immunoprecipitated with N30 antibody and analyzed by IB using monoclonal anti-PRLr antibody from Invitrogen. NS, Nonspecific band. C, Lysates from the T47D cells (treated with or without 100 ng/ml of PRL for 15 min as indicated) were immunoprecipitated using either NRS or anti-PRLr N30 polyclonal antibody. Levels of PRLr and PKM2 in these reactions were detected by IB as indicated. Levels of PKM2, PRLr, and total and phosphorylated in the WCEs are also shown. IgG, Immunoglobulin heavy chain.

Article Snippet: Conversely, PRLr immunoprecipitated with N30 antibody was detected by commercially available monoclonal anti-PRLr antibody (Fig. 2B ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 A, Immunoprecipitation (IP) of endogenous PRLr from lysates from 293T cells treated with or without human PRL (purchased from the National Hormone and Peptide program and used at 100 ng/ml for 30 min) was carried out using anti-PRLr antibody (H-300, Santa Cruz) or naïve rabbit serum (NRS).

Techniques: Immunoprecipitation, Infection, SDS Page, Western Blot

Journal:

Article Title: Prolactin Inhibits Activity of Pyruvate Kinase M2 to Stimulate Cell Proliferation

doi: 10.1210/me.2010-0219

Figure Lengend Snippet: A, Pyruvate kinase activity was determined in lysates (50 μg) from 293T cells treated with PRL (100 ng/ml for indicated time points) by a coupled enzymatic-based spectrophotometric assay as described in Materials and Methods. Average data from four independent experiments (each in triplicate) are presented as percent of activity measured in cells that did not receive PRL (±sd). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). B, Transient expression of indicated Flag-tagged PRLr species in 293T cells was verified by immunoblotting (IB) using indicated antibodies. C, Activity of pyruvate kinase was determined (as in panel A) in lysates from 293T cells transfected with vector control (pCDNA3) or vectors for expression of PRLr [wild type (WT) or S349A mutant] and treated (100 ng/ml PRL for 20 min, white bars) or not (gray bars) with PRL. Here and in similar subsequent figures, the average data from three independent experiments (each in triplicate) are presented as percent of activity of nontreated control cells (±sd). Absolute values of PKM activity in untreated cells transfected with different PRLr constructs were comparable (data not shown). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). D, Pyruvate kinase activity was measured in the lysates from MCF10a-derived cells expressing wild-type or S349A mutant of PRLr (described in details in Ref. 12) as in panel B. E, Lactate levels in the lysates from cells used in panel C were determined using a fluorescence-based lactate measurement assay. Asterisks signify that the difference in lactate levels between the treated and untreated samples is significant as determined by Student’s t test (P < 0.05).

Article Snippet: Conversely, PRLr immunoprecipitated with N30 antibody was detected by commercially available monoclonal anti-PRLr antibody (Fig. 2B ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 A, Immunoprecipitation (IP) of endogenous PRLr from lysates from 293T cells treated with or without human PRL (purchased from the National Hormone and Peptide program and used at 100 ng/ml for 30 min) was carried out using anti-PRLr antibody (H-300, Santa Cruz) or naïve rabbit serum (NRS).

Techniques: Activity Assay, Spectrophotometric Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Mutagenesis, Construct, Derivative Assay, Fluorescence, Lactate Assay

Journal:

Article Title: Prolactin Inhibits Activity of Pyruvate Kinase M2 to Stimulate Cell Proliferation

doi: 10.1210/me.2010-0219

Figure Lengend Snippet: A, Levels of endogenous PRLr in T47D-derived cells that harbor shCON or shRNA against PRLr (shPRLr) were determined by immunoprecipitation (IP)-immunoblotting (IB) as described in details in Ref. 12. B, Pyruvate kinase activity was measured in the lysates from T47D-derived cells that harbor shCON or shRNA against PRLr (shPRLr). Cells (that were described in details in Ref. 12) were left untreated (gray bars) or treated with PRL (100 ng/ml for 20 min, white bars). Absolute values of PKM activity in untreated cells transfected with different shRNA constructs were comparable (data not shown). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). C, Lactate levels in the lysates from cells used in panel B were determined as outlined in Fig. 3E​3E.

Article Snippet: Conversely, PRLr immunoprecipitated with N30 antibody was detected by commercially available monoclonal anti-PRLr antibody (Fig. 2B ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 A, Immunoprecipitation (IP) of endogenous PRLr from lysates from 293T cells treated with or without human PRL (purchased from the National Hormone and Peptide program and used at 100 ng/ml for 30 min) was carried out using anti-PRLr antibody (H-300, Santa Cruz) or naïve rabbit serum (NRS).

Techniques: Derivative Assay, shRNA, Immunoprecipitation, Western Blot, Activity Assay, Transfection, Construct

Journal:

Article Title: Prolactin Inhibits Activity of Pyruvate Kinase M2 to Stimulate Cell Proliferation

doi: 10.1210/me.2010-0219

Figure Lengend Snippet: A, Transient expression of indicated hemaggltinin (HA)-tagged and V5-tagged PRLr species in 293T cells was verified by immunoblotting (IB) using indicated antibodies. B, Activity of pyruvate kinase (PK) was determined (as in Fig. 3A​3A)) in lysates from 293T cells transfected with vector control (pCDNA3) or vectors for expression of PRLr [wild type or YF mutant (all intracellular tyrosines mutated to phenylalanine, described in Ref. 16)] and treated (100 ng/ml PRL for 20 min, white bars) or not (gray bars) with PRL. Absolute values of PKM activity in untreated cells transfected with either PRLrWT or PRLrYF were comparable (data not shown). Average data from four independent experiments (each in triplicate) are presented as percent of activity measured in cells that did not receive PRL (± sd). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). C, Lactate levels in the lysates from cells used in panel B were determined as outlined in Fig. 3E​3E.. D, PK activity was determined in lysates from 293T cells transfected with vector control (pCDNA3) or vectors for expression of PRLr (wild-type or I170L mutant). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). E, Lactate levels in the lysates from cells used in panel D were determined as outlined in Fig. 3E​3E.

Article Snippet: Conversely, PRLr immunoprecipitated with N30 antibody was detected by commercially available monoclonal anti-PRLr antibody (Fig. 2B ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 A, Immunoprecipitation (IP) of endogenous PRLr from lysates from 293T cells treated with or without human PRL (purchased from the National Hormone and Peptide program and used at 100 ng/ml for 30 min) was carried out using anti-PRLr antibody (H-300, Santa Cruz) or naïve rabbit serum (NRS).

Techniques: Expressing, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Control, Mutagenesis