Journal: Cell reports

Article Title: Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation.

doi: 10.1016/j.celrep.2016.04.066

Figure Lengend Snippet: Figure 6. PD407824 Increases BMP Sensitivity by Depleting p21 and Activating CDK9, which Then Phosphorylates the SMAD2/3 Linker Region, Leading to Decreased Levels of SMAD2/3 Protein and Enhanced Levels of Nuclear SMAD1 (A) PD407824 treatment increases the binding of SMAD1 and SMAD4 proteins indicated by co-immunoprecipitation. (B) PD407824 causes depletion of SMAD2/3 protein levels, measured by western blotting; SMAD2/3 protein levels are normalized to beta-ACTIN protein levels. (C) PD407824 induces the phosphorylation of the SMAD2/3 linker region (T179 for SMAD3). The phosphorylation level was normalized to total levels of SMAD2 protein. (D–F) Flavopiridol, a CDK inhibitor, blocks the PD407824 induced increased Id2 transcript expression (D), upregulated T179 phosphorylation of SMAD3 and decreased SMAD2/3 total protein (E), and increased binding of SMAD1 and SMAD4 proteins indicated by co-immunoprecipitation (F). (G) Knockout of CDK9 blocks the synergistic effect of PD407824. (H) PD407824 depletes p21 protein levels, shown by western blotting. The p21 protein levels are normalized to b-actin protein levels. (I) Relative Id2 transcript expression in p21+/ and p21/ cells. Uninfected cells (wild-type [wt]) and cells infected with sgRNA targeting GFP were used as negative controls. See also Figure S4.

Article Snippet: The antibodies were rabbit anti-phospho-SMAD1/5/9 (Cell Signaling Technology, 9511), rabbit anti-SMAD1 XP (Cell Signal, 6944), rabbit anti-SMAD2/3 (Cell Signal, 3102), rabbit anti-p21 (C-19 antibody; Santa Cruz Biotechnology, sc379), rabbit anti-phospho-SMAD3 T179 (Rockland, 600-401-C48S), and mouse anti-b-actin (Sigma-Aldrich, A1978).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, Expressing, Knock-Out, Infection

Journal: Cell Death & Disease

Article Title: ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance

doi: 10.1038/s41419-024-06647-0

Figure Lengend Snippet: a Dephosphorylation of ELK3 prevent the interaction between ELK3 and SPOP. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were treated with/without λ-phosphatase. The interaction between ELK3 and SPOP was evaluated by IP and WB. b CHK1/2 inhibition suppresses ELK3 destabilization. The cell lysates of HeLa cells treated with 5 μM of indicated inhibitor and CHX (10 μg/ml) for 12 h were used to evaluate the ELK3 protein levels by WB. c CHK1/2 inhibition abolishes ELK3 and SPOP interaction. The cell lysates of HEK293T cells transfected with indicated plasmids treated with 5 μM of indicated inhibitor for 12 h and MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and SPOP by IP and WB. d ELK3 interacts with CHK1 and CHK2. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and CHK1 or CHK2 by IP and WB. e CHK2 facilitates ELK3 ubiquitination by SPOP. cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. f CHK1/2 inhibition abrogates SPOP-mediated ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with 5 μM of AZD7762 for 12 h and MG132 (10 μM) for 4 h were used to evaluate the SPOP-mediated ELK3 ubiquitination by IP and WB. g CHK1 knockdown increases ELK3 protein levels. The cell lysates of HeLa cells stably expressing sh-mock or sh-CHK1 or CHK2 were used to evaluate ELK3 protein levels by WB. h CHK1 and CHK2 phosphorylates ELK3. In vitro kinase assay using partially purified His-ELK3 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. i ELK3 deg1 deletion abolishes CHK1- or CHK2-mediated phosphorylation. In vitro kinase assay using partial purified His-ELK3-wt or -∆Deg1 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. j , k CHK2 phosphorylates ELK3 at Ser133 in cell system. The phosphorylation of ELK3 mediated by CHK2 in cell culture ( j ) and in vitro kinase assay system ( k ) was evaluated using phos-tag immunoblot analysis. Treatment with AZD7762 ( j ) and mutation of ELK3 Ser133 to Ala ( l ) abolished the phosphorylation of ELK3 induced by CHK2. l ELK3 phosphorylation at Ser133 is indispensable to interact with SPOP. ELK3 phosphorylation requirement for the interaction with SPOP was evaluated by IP and western blotting using cell lysates transiently expressing mock, His-ELK3-wt, His-ELK3-S133D, or His-ELK3-S133A.

Article Snippet: Subsequently, the partially purified ELK3-wt (1 μg) and ELK3-ΔDeg1 (1 μg) were combined with active CHK1 (cat. no.: C47-10H, SignalChem, Richmond, BC, Canada) and CHK2 (cat. no.: C48-10G, SignalChem) and cold ATP.

Techniques: De-Phosphorylation Assay, Transfection, Inhibition, Ubiquitin Proteomics, Knockdown, Stable Transfection, Expressing, In Vitro, Kinase Assay, Purification, Phospho-proteomics, Cell Culture, Western Blot, Mutagenesis

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: c48/80- and LPA-stimulated G protein activity in brain cryostat sections is mediated by LPA receptors, likely LPA1. (a) [35S]GTPγS autoradiography was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. c48/80 (100 μg ml−1; MP Biomedicals) or LPA (5 μM in 0.1% fatty acid-free BSA) was included in the [35S]GTPγS labeling step of developing (4-week-old) rat brain sections along with or without the LPA1/LPA3 receptor-selective antagonist Ki16425 (5 μM). Note that Ki16425 clearly abolishes [35S]GTPγS binding responses to c48/80 and LPA throughout the white matter tracts. Like 1-butanol, Ki16425 also suppresses basal G protein activity in the white matter regions, indicating tonic LPA receptor activity in the developing rat brain. Abbreviations: cca, corpus callosum. Scale bar=5 mm. (b) Quantitative autoradiography data on the corpus callosum, selected to represent the white matter regions. Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images for statistical analysis. Ki16425 dose-dependently decreases the basal [35S]GTPγS binding as well as that evoked by c48/80 (100 μg ml−1; MP Biomedicals) or LPA (5 μM) with IC50 values of 35±9, 59±59 and 87±19 nM, respectively. Values are mean±s.e.m. (or IC50±s.e.m.) representing six sections from six developing (4-week-old) animals.

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Activity Assay, Autoradiography, Labeling, Binding Assay, Radioactivity

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: [35S]GTPγS autoradiography of rat brain sections reveals LPA-mimicking and NEM-sensitive G protein activation in response to stimulation with c48/80. (a) [35S]GTPγS autoradiography was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. c48/80 (100 μg ml−1) from two suppliers (MP Biomedicals and Sigma-Aldrich) or LPA (50 μM in 0.1% fatty acid-free BSA) was present during the [35S]GTPγS labeling in step 3. Some sections were treated with the irreversible Gi/o protein inhibitor NEM (1 mM, included in step 1 of the protocol). In the control panel (left), the white matter anatomical loci where LPA typically activates G proteins are indicated. Note the highly restricted and LPA-mimicking distribution of c48/80-stimulated G protein activity throughout the white matter tracts, including the anterior commissure (aca), the corpus callosum (cca) and the fimbria of the hippocampus (fi). Other abbreviations: ctx, cerebral cortex; hip, hippocampal structures; str, striatum. Scale bar=5 mm. (b) Quantitative autoradiography data on selected brain regions and on whole brain for NEM-pretreated sections (inset). Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images for two white matter regions (corpus callosum (cca) and the fimbria of the hippocampus (fi)), two gray matter regions (cerebral cortex (ctx) and hippocampus (hip)), an area containing both white and gray matter (striatum (str)) and for whole section area, as detailed in the Methods section. Values are mean+s.e.m. representing sections from four individual animals. Risk level: *P<0.05 compared to basal in each specified brain region and #P<0.05 compared to NEM-pretreated basal on whole section radioactivity (one-way ANOVA with Tukey's multiple comparison).

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Autoradiography, Activation Assay, Labeling, Control, Activity Assay, Radioactivity, Comparison

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: c48/80 stimulates G protein activity in the developing white matter tracts through 1-butanol-sensitive mechanisms likely involving PLD. (a) [35S]GTPγS autoradiography of developing (4-week-old) and adult (9-week-old) rat brain sections was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. c48/80 (100 μg ml−1; MP Biomedicals) or LPA (50 μM in 0.1% fatty acid-free BSA) and the structural butanol isomers (2%, vol vol−1) were present during the [35S]GTPγS labeling step, as indicated. Note that the PLD inhibitor 1-butanol but not its inactive isomer tert-butanol selectively inhibits c48/80-stimulated G protein activity in the developing white matter tracts without affecting LPA-evoked responses. Note also age-dependent decline in basal and c48/80- or LPA-evoked [35S]GTPγS binding responses throughout the adult white matter tracts. Abbreviations: cca, corpus callosum; fi; the fimbria of the hippocampus; cbw, the cerebellar white matter (cbw). Scale bar=5 mm. (b) Quantitative autoradiography data on the corpus callosum of 4-week-old (left) and 9-week-old rats (right). Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images, as detailed in the Methods section. Values are mean+s.e.m. representing four sections from four (developing) or two (adult) individual animals. Risk level: *P<0.05 compared to the respective control treatment in the corpus callosum of 4-week-old rat (one-way ANOVA with Tukey's multiple comparison).

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Activity Assay, Autoradiography, Labeling, Binding Assay, Radioactivity, Control, Comparison

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: (a) Known phospholipase 2 (PLA2) inhibitors, bromoenol lactone (BEL, calcium-independent PLA2), arachidonoyltrifluoromethyl ketone (ATFMK, cytosolic PLA2) and p-bromophenacyl bromide (BPB, secretatory PLA2), have no effect on the c48/80-induced [35S]GTPγS binding to 4-week-old rat brain white matter areas. [35S]GTPγS autoradiography was conducted in three steps with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. (b) Quantitative autoradiography data on the corpus callosum, selected to represent the white matter. The autoradiography images were digitized and bound radioactivity values were obtained from the digitized images for statistical analysis. Values are mean+s.e.m. representing four sections from four developing (4-week-old) animals. Scale bar=5 mm. c48/80 (100 μg ml−1; MP Biomedicals) was included in the labeling step of autoradiography along with the inhibitors (each at 50 μM) as indicated in the figure. No significant alterations were detected in c48/80-induced [35S]GTPγS binding when inhibitors were included. Risk level: P<0.05 compared to the respective control treatment in the corpus callosum.

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Binding Assay, Autoradiography, Radioactivity, Labeling, Control

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: In alkaline conditions, c48/80 activates rat brain heterotrimeric G proteins through the 5-HT1A receptors. (a) [35S]GTPγS autoradiography was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section except that buffer pH was 8.40. At 100 μg ml−1, c48/80 stimulates [35S]GTPγS binding to the hippocampal structures (hip), identical to those labeled by the selective 5-HT1A receptor agonist 8-OH-DPAT (1 μM). Both c48/80- and 8-OH-DPAT-evoked responses are reversed by the selective 5-HT1A receptor antagonist NAN-190 (1 μM). Scale bar=5 mm. (b) Quantitative autoradiography data on the hippocampal region. Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images, as detailed in the Methods section. Values are mean+s.e.m. representing sections from four individual animals. Risk level: *P<0.05 as compared to the basal and #P<0.05 as compared to the 8-OH-DPAT- or c48/80-induced response in the absence of NAN-190 (one-way ANOVA with Tukey's multiple comparison).

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Autoradiography, Binding Assay, Labeling, Radioactivity, Comparison

Journal:

Article Title: The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT 1A receptors in rat brain sections

doi: 10.1038/sj.bjp.0706671

Figure Lengend Snippet: c48/80, unlike MP-7 or stimulation of G protein-coupled muscarinic acetylcholine receptors, fails to enhance the rate of [35S]GTPγS binding to rat forebrain membranes. Certain commercial batches of c48/80 were also found unsuitable for [35S]GTPγS-based G protein activation assays. (a) Classical [35S]GTPγS membrane binding assay was conducted, as detailed in the Methods section. Rat forebrain membranes were incubated for 90 min together with 100 μg ml−1 c48/80 from MP Biomedicals or Sigma-Aldrich in the presence of 0.15 nM [35S]GTPγS with or without 10 μM excess of non-labeled GTPγS. The data represent the percentage of bound radioactivity+s.e.m. from three independent experiments performed in duplicate. A batch of c48/80 from Sigma-Aldrich, but not from MP Biomedicals, causes a significant amount of [35S]GTPγS labeling, even when excess of non-labeled GTPγS is present, probably indicating, for example, chemical interaction between the guanine nucleotides and some portion of the c48/80 mixture. *P<0.05 compared to basal bound radioactivity at 90 min and #P<0.05 compared to Nsb in basal conditions. (b) Rat forebrain membranes were incubated in the presence of c48/80 (100 μg ml−1; MP Biomedicals), MP-7 (50 μM) or carbachol (100 μM) for the indicated times. Data are represented as percentage of bound radioactivity±s.e.m. from three independent experiments performed in duplicate. At all time points tested, MP-7 and carbachol, but not c48/80, significantly stimulated [35S]GTPγS labeling. Risk level: P<0.05 compared to basal condition in each time point.

Article Snippet: Another batch of c48/80 from Sigma-Aldrich likewise increased [ 35 S]GTP γ S binding in both white and gray matter brain regions, whereas c48/80 obtained from Biomol, like the one from MP Biomedicals, stimulated [ 35 S]GTP γ S binding specifically to the white matter tracts (data not shown).

Techniques: Binding Assay, Activation Assay, Membrane, Incubation, Labeling, Radioactivity

Journal: Frontiers in Microbiology

Article Title: Desirable Traits of a Good Biocontrol Agent against Verticillium Wilt

doi: 10.3389/fmicb.2017.01186

Figure Lengend Snippet: Bacterial isolates with biocontrol activity against Verticillium in different host plants.

Article Snippet: Seed treatment with S. plymuthica HRO-C48 could also protect cotton plants against Verticillium wilt (Erdogan and Benlioglu, ).

Techniques: Activity Assay

Journal: BMC Microbiology

Article Title: Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption

doi: 10.1186/1471-2180-9-251

Figure Lengend Snippet: Reference, probiotic and faecal LAB isolates examined or isolated during the study

Article Snippet: C48 ( EF442283 ) , L. paracasei subsp. paracasei DJ1 ( DQ462440 ) , Cultech Ltd. , 11.

Techniques: Isolation, Sequencing

Journal: BMC Microbiology

Article Title: Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption

doi: 10.1186/1471-2180-9-251

Figure Lengend Snippet: Discrimination of LAB by RAPD typing . The ability of PCR fingerprinting (with primer 272) to cluster identical isolates (Panel A) and differentiate distinct isolates within the L. casei group (Panel B) is shown. Strains shown in each lane are as follows: Panel A; 1, L. acidophilus LMG 9433 T ; lanes 2 to 6, matching L. acidophilus isolates LMG 11428, LMG 11430, C21, C46 and NCIMB 30211, respectively; Panel B; lanes 7 to 11, L. paracasei subsp paracasei isolates C48, C65, C83, C79 and LMG 7955, respectively; 12, L. casei LMG 6904 T ; and 13, L. rhamnosus MW. Molecular size markers were run in lane M and the size of relevant bands is indicated; panel A and B represent composite lanes taken from a single gel in each case.

Article Snippet: C48 ( EF442283 ) , L. paracasei subsp. paracasei DJ1 ( DQ462440 ) , Cultech Ltd. , 11.

Techniques:

Journal: Oncotarget

Article Title: An extract from the Atlantic brown algae Saccorhiza polyschides counteracts diet-induced obesity in mice via a gut related multi-factorial mechanisms

doi: 10.18632/oncotarget.18113

Figure Lengend Snippet: Food intake and fecal excretion is increased in high fat diet fed mice supplemented with a brown algae extract (BAE)

Article Snippet: Fecal caloric value was determined with a bomb calorimeter (C 7000, cooler C7002, oxygen filling station C48, IKA ® -Werke GmbH & Co. KG, Staufen, Germany) and apparent food digestibility was calculated as (total energy intake - fecal energy excretion)/total energy intake*100.

Techniques: Algae