Structured Review

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Chloroquine inhibits degradation of HIF1a in non-melanoma but not in melanoma cells. (A and B) PC3 (prostate cancer) and HT1080 (osteosarcoma) cell lines were treated with 50 µM chloroquine (CQ) or 100 µM deferoxamine (DFO) for 8 h. Cell extracts (20 µg/sample) were resolved on a SDS-PAGE and blotted with HIF1a, <t>LC3</t> and tubulin antibodies. (C, D and E) MEL526, RPMI8322, MEL2664 melanoma lines cell lines exposed to CQ or DFO and cell lysates were probed with indicated antibodies. (F and G) MEL526 and PC3 cell lines were exposed to CQ (50 µM), DMOG (500 µM), DFO (100 µM), MLN4924 (10 µM) and MG132 (5 µM) for 8 h and cell lysates were blotted with HIF1a, LC3 and tubulin antibodies.
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1) Product Images from "Golgi Associated HIF1a Serves as a Reserve in Melanoma Cells"

Article Title: Golgi Associated HIF1a Serves as a Reserve in Melanoma Cells

Journal: Journal of cellular biochemistry

doi: 10.1002/jcb.25381

Chloroquine inhibits degradation of HIF1a in non-melanoma but not in melanoma cells. (A and B) PC3 (prostate cancer) and HT1080 (osteosarcoma) cell lines were treated with 50 µM chloroquine (CQ) or 100 µM deferoxamine (DFO) for 8 h. Cell extracts (20 µg/sample) were resolved on a SDS-PAGE and blotted with HIF1a, LC3 and tubulin antibodies. (C, D and E) MEL526, RPMI8322, MEL2664 melanoma lines cell lines exposed to CQ or DFO and cell lysates were probed with indicated antibodies. (F and G) MEL526 and PC3 cell lines were exposed to CQ (50 µM), DMOG (500 µM), DFO (100 µM), MLN4924 (10 µM) and MG132 (5 µM) for 8 h and cell lysates were blotted with HIF1a, LC3 and tubulin antibodies.
Figure Legend Snippet: Chloroquine inhibits degradation of HIF1a in non-melanoma but not in melanoma cells. (A and B) PC3 (prostate cancer) and HT1080 (osteosarcoma) cell lines were treated with 50 µM chloroquine (CQ) or 100 µM deferoxamine (DFO) for 8 h. Cell extracts (20 µg/sample) were resolved on a SDS-PAGE and blotted with HIF1a, LC3 and tubulin antibodies. (C, D and E) MEL526, RPMI8322, MEL2664 melanoma lines cell lines exposed to CQ or DFO and cell lysates were probed with indicated antibodies. (F and G) MEL526 and PC3 cell lines were exposed to CQ (50 µM), DMOG (500 µM), DFO (100 µM), MLN4924 (10 µM) and MG132 (5 µM) for 8 h and cell lysates were blotted with HIF1a, LC3 and tubulin antibodies.

Techniques Used: SDS Page

2) Product Images from "Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding"

Article Title: Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding

Journal: The EMBO Journal

doi: 10.15252/embj.201593378

KJE acts prior to the disaggregation step of IbpAB–luciferase assemblies An order of addition experiment. Luciferase (1.5 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM), diluted to 40 nM and incubated first for 20 min with the components listed in the legend and then complemented with the missing components of the functional ClpB‐KJE bichaperone system. The DnaK, DnaJ, GrpE, ClpB concentrations used were as follows: 1, 0.3, 0.3 and 1.5 μM. Data are the mean ± SD of three independent experiments.
Figure Legend Snippet: KJE acts prior to the disaggregation step of IbpAB–luciferase assemblies An order of addition experiment. Luciferase (1.5 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM), diluted to 40 nM and incubated first for 20 min with the components listed in the legend and then complemented with the missing components of the functional ClpB‐KJE bichaperone system. The DnaK, DnaJ, GrpE, ClpB concentrations used were as follows: 1, 0.3, 0.3 and 1.5 μM. Data are the mean ± SD of three independent experiments.

Techniques Used: Luciferase, Incubation, Functional Assay

Hsp70, independently of Hsp100, releases IbpAB‐dependent inhibition of disaggregation IbpAB association with GFP aggregates blocks substrate translocation through the central Hsp100 pore. Aggregated GFP (1.5 μM) was incubated for 10 min at 48°C (marked “T” in the Figure) alone (blue) or with IbpA and IbpB (3 μM and 7 μM, respectively; sky blue) or aggregated GFP and IbpAB were incubated separately at 48°C and then mixed (orange). Hsp104 D484K hyperactive variant (1 μM) was added, and GFP reactivation was monitored. KJE machinery allows to overcome the IbpAB‐dependent inhibition of IbpAB–GFP disaggregation by hyperactive Hsp104 D484K. Aggregated GFP was incubated alone at 48°C (blue and orange) or with IbpAB at 48°C (sky blue and black). After addition of the Hsp104 D484K hyperactive mutant (all plots) and KJE machinery (black and magenta), GFP reactivation was monitored. DnaK, DnaJ and GrpE were present at 1 μM, 0.3 μM and 0.3 μM. Aggregated GFP, IbpAB and Hsp104 D484K were in concentrations as in (A).
Figure Legend Snippet: Hsp70, independently of Hsp100, releases IbpAB‐dependent inhibition of disaggregation IbpAB association with GFP aggregates blocks substrate translocation through the central Hsp100 pore. Aggregated GFP (1.5 μM) was incubated for 10 min at 48°C (marked “T” in the Figure) alone (blue) or with IbpA and IbpB (3 μM and 7 μM, respectively; sky blue) or aggregated GFP and IbpAB were incubated separately at 48°C and then mixed (orange). Hsp104 D484K hyperactive variant (1 μM) was added, and GFP reactivation was monitored. KJE machinery allows to overcome the IbpAB‐dependent inhibition of IbpAB–GFP disaggregation by hyperactive Hsp104 D484K. Aggregated GFP was incubated alone at 48°C (blue and orange) or with IbpAB at 48°C (sky blue and black). After addition of the Hsp104 D484K hyperactive mutant (all plots) and KJE machinery (black and magenta), GFP reactivation was monitored. DnaK, DnaJ and GrpE were present at 1 μM, 0.3 μM and 0.3 μM. Aggregated GFP, IbpAB and Hsp104 D484K were in concentrations as in (A).

Techniques Used: Inhibition, Translocation Assay, Incubation, Variant Assay, Mutagenesis

KJE dissociates IbpAB from IbpAB–MDH assemblies in vitro Experimental scheme. IbpAB–MDH assemblies, isolated from unbound IbpAB via sedimentation in glycerol gradient, were incubated with the indicated components for 120 min and resubjected to a second round of sedimentation. KJE dissociates IbpAB from IbpAB–MDH assemblies. Fractions were collected from the top of the gradient, and MDH and IbpA were visualized by Western blot following SDS–PAGE. IbpA and MDH levels in each fraction were determined by quantitative immunoblot analysis using IbpA‐ and MDH‐specific antibodies, respectively. The band intensities of IbpA and MDH retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and plotted on the graphs on the left. IbpAB–MDH assemblies (approximate stoichiometry 1:0.5:1), DnaK, DnaJ, GrpE and ClpB were present at 400 nM (calculated for MDH monomers), 1 μM, 0.3 μM, 0.3 μM and 1.5 μM concentrations, respectively. Source data are available online for this figure.
Figure Legend Snippet: KJE dissociates IbpAB from IbpAB–MDH assemblies in vitro Experimental scheme. IbpAB–MDH assemblies, isolated from unbound IbpAB via sedimentation in glycerol gradient, were incubated with the indicated components for 120 min and resubjected to a second round of sedimentation. KJE dissociates IbpAB from IbpAB–MDH assemblies. Fractions were collected from the top of the gradient, and MDH and IbpA were visualized by Western blot following SDS–PAGE. IbpA and MDH levels in each fraction were determined by quantitative immunoblot analysis using IbpA‐ and MDH‐specific antibodies, respectively. The band intensities of IbpA and MDH retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and plotted on the graphs on the left. IbpAB–MDH assemblies (approximate stoichiometry 1:0.5:1), DnaK, DnaJ, GrpE and ClpB were present at 400 nM (calculated for MDH monomers), 1 μM, 0.3 μM, 0.3 μM and 1.5 μM concentrations, respectively. Source data are available online for this figure.

Techniques Used: In Vitro, Isolation, Sedimentation, Incubation, Western Blot, SDS Page

Hsp70 releases sHsps from the fraction of aggregated proteins in vivo Experimental scheme. BB6410 (PA1lacO‐1) dnaK, dnaJ, lacIq , Δ clpB cells were grown at 30°C in LB medium until late log phase, heat‐shocked at 46°C for 30 min, then the culture was separated into two flasks and IPTG was added at 1 mM. At indicated time points, aliquots were taken, cells were lysed and protein aggregates were isolated. The isolated fraction of aggregates was analysed by SDS–PAGE followed by staining with Coomassie brilliant blue at the indicated time points. Levels of IbpA present in aggregates at the indicated time points were determined by quantitative immunoblot analysis using IbpA‐specific antibodies. Quantification of (B) and (C). DnaK cellular levels were determined by quantitative immunoblot analysis using DnaK‐specific antibodies. Source data are available online for this figure.
Figure Legend Snippet: Hsp70 releases sHsps from the fraction of aggregated proteins in vivo Experimental scheme. BB6410 (PA1lacO‐1) dnaK, dnaJ, lacIq , Δ clpB cells were grown at 30°C in LB medium until late log phase, heat‐shocked at 46°C for 30 min, then the culture was separated into two flasks and IPTG was added at 1 mM. At indicated time points, aliquots were taken, cells were lysed and protein aggregates were isolated. The isolated fraction of aggregates was analysed by SDS–PAGE followed by staining with Coomassie brilliant blue at the indicated time points. Levels of IbpA present in aggregates at the indicated time points were determined by quantitative immunoblot analysis using IbpA‐specific antibodies. Quantification of (B) and (C). DnaK cellular levels were determined by quantitative immunoblot analysis using DnaK‐specific antibodies. Source data are available online for this figure.

Techniques Used: In Vivo, Isolation, SDS Page, Staining

KJE acts prior to disaggregation of IbpAB–MDH assemblies An order of addition experiment. MDH (2 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM) at 47°C for 30 min. IbpAB–MDH assemblies were diluted fourfold and first incubated with the components listed in the legend and then complemented with the missing components of the functional ClpB‐KJE bichaperone system. The DnaK, DnaJ, GrpE, ClpB concentrations used were as follows: 1, 0.3, 0.3 and 1.5 μM. Data are the mean ± SD of three independent experiments.
Figure Legend Snippet: KJE acts prior to disaggregation of IbpAB–MDH assemblies An order of addition experiment. MDH (2 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM) at 47°C for 30 min. IbpAB–MDH assemblies were diluted fourfold and first incubated with the components listed in the legend and then complemented with the missing components of the functional ClpB‐KJE bichaperone system. The DnaK, DnaJ, GrpE, ClpB concentrations used were as follows: 1, 0.3, 0.3 and 1.5 μM. Data are the mean ± SD of three independent experiments.

Techniques Used: Incubation, Functional Assay

IbpAB–luciferase assemblies differ in biophysical and biochemical properties from luciferase aggregates A Dynamic light scattering graphs depicting the size distribution of the following entities: native luciferase (green), IbpAB (grey), luciferase aggregates (red), IbpAB–luciferase assemblies (blue), shown as volume distribution of analysed species. Luciferase was present at 1.5 μM, IbpA at 3 μM, IbpB at 7 μM concentration. B Scheme of the disaggregation experiment. C, D Renaturation kinetics for luciferase denatured in the presence or absence of IbpAB. Luciferase (1.5 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM), diluted to 40 nM and refolded at limited (0.3 μM; C) or elevated (2.4 μM; D) KJE machinery concentrations and standard (1.5 μM) ClpB concentration. The KJE machinery concentration is given with respect to DnaK with a constant DnaK:DnaJ:GrpE ratio 1:0.3:0.3. Data are the mean ± SD of three independent experiments. E A comparison of renaturation rates for luciferase denatured in the presence or absence of IbpAB in changing KJE and constant ClpB concentrations. The KJE machinery concentration is given with respect to DnaK with a constant DnaK:DnaJ:GrpE ratio 1:0.3:0.3. F A comparison of renaturation rates for luciferase denatured in the presence or absence of IbpAB in changing ClpB and constant KJE concentrations. The dashed line indicates the theoretical scenario that luciferase refolding from aggregates and sHsp assemblies proceeds with identical refolding rates.
Figure Legend Snippet: IbpAB–luciferase assemblies differ in biophysical and biochemical properties from luciferase aggregates A Dynamic light scattering graphs depicting the size distribution of the following entities: native luciferase (green), IbpAB (grey), luciferase aggregates (red), IbpAB–luciferase assemblies (blue), shown as volume distribution of analysed species. Luciferase was present at 1.5 μM, IbpA at 3 μM, IbpB at 7 μM concentration. B Scheme of the disaggregation experiment. C, D Renaturation kinetics for luciferase denatured in the presence or absence of IbpAB. Luciferase (1.5 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM), diluted to 40 nM and refolded at limited (0.3 μM; C) or elevated (2.4 μM; D) KJE machinery concentrations and standard (1.5 μM) ClpB concentration. The KJE machinery concentration is given with respect to DnaK with a constant DnaK:DnaJ:GrpE ratio 1:0.3:0.3. Data are the mean ± SD of three independent experiments. E A comparison of renaturation rates for luciferase denatured in the presence or absence of IbpAB in changing KJE and constant ClpB concentrations. The KJE machinery concentration is given with respect to DnaK with a constant DnaK:DnaJ:GrpE ratio 1:0.3:0.3. F A comparison of renaturation rates for luciferase denatured in the presence or absence of IbpAB in changing ClpB and constant KJE concentrations. The dashed line indicates the theoretical scenario that luciferase refolding from aggregates and sHsp assemblies proceeds with identical refolding rates.

Techniques Used: Luciferase, Concentration Assay

Hsp70 releases sHsps from sHsp–substrate assemblies in vitro Experimental scheme. Purified IbpAB–luciferase assemblies were incubated with indicated components for the indicated time and resubjected to glycerol gradient sedimentation. KJE releases IbpAB from IbpAB–luciferase assemblies. Fractions were collected from the top of the gradient, and luciferase and IbpA were visualized by Western blot following SDS–PAGE. The band intensities of luciferase and IbpA retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and plotted on the graphs on the left. IbpA–IbpB–luciferase assemblies (approximate stoichiometry 1:0.8:1), DnaK, DnaJ and GrpE were present at 100 nM (calculated for luciferase monomer), 1 μM, 0.3 μM and 0.3 μM concentrations, respectively. Concerted action of ClpB‐KJE results in release of IbpAB and luciferase from IbpAB–luciferase assemblies. Experiments were performed and analysed as in (B). ClpB was present at 1.5 μM concentration. Source data are available online for this figure.
Figure Legend Snippet: Hsp70 releases sHsps from sHsp–substrate assemblies in vitro Experimental scheme. Purified IbpAB–luciferase assemblies were incubated with indicated components for the indicated time and resubjected to glycerol gradient sedimentation. KJE releases IbpAB from IbpAB–luciferase assemblies. Fractions were collected from the top of the gradient, and luciferase and IbpA were visualized by Western blot following SDS–PAGE. The band intensities of luciferase and IbpA retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and plotted on the graphs on the left. IbpA–IbpB–luciferase assemblies (approximate stoichiometry 1:0.8:1), DnaK, DnaJ and GrpE were present at 100 nM (calculated for luciferase monomer), 1 μM, 0.3 μM and 0.3 μM concentrations, respectively. Concerted action of ClpB‐KJE results in release of IbpAB and luciferase from IbpAB–luciferase assemblies. Experiments were performed and analysed as in (B). ClpB was present at 1.5 μM concentration. Source data are available online for this figure.

Techniques Used: In Vitro, Purification, Luciferase, Incubation, Sedimentation, Western Blot, SDS Page, Concentration Assay

Prokaryotic Hsp70 dissociates eukaryotic sHsps from sHsp–substrate assemblies in vitro Experimental scheme. Hsp26–MDH assemblies isolated from unbound Hsp26 via sedimentation in a glycerol gradient were incubated with the indicated components for 120 min and resubjected to a second round of sedimentation. DnaK, DnaJ, GrpE dissociate Hsp26 from Hsp26–MDH assemblies. Fractions were collected from the top of the gradient and MDH and Hsp26 were visualized by Western blot following SDS–PAGE. MDH and Hsp26 levels in each fraction were determined by quantitative immunoblot analysis using MDH‐ and Hsp26‐specific antibodies, respectively. The band intensities of Hsp26 and MDH retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and presented on the graphs on the left. Hsp26–MDH assemblies (approximate stoichiometry 1.25:1), DnaK, DnaJ, GrpE and ClpB were present at 400 nM (calculated for MDH monomers), 1 μM, 0.3 μM, 0.3 μM and 1.5 μM concentrations, respectively. Source data are available online for this figure.
Figure Legend Snippet: Prokaryotic Hsp70 dissociates eukaryotic sHsps from sHsp–substrate assemblies in vitro Experimental scheme. Hsp26–MDH assemblies isolated from unbound Hsp26 via sedimentation in a glycerol gradient were incubated with the indicated components for 120 min and resubjected to a second round of sedimentation. DnaK, DnaJ, GrpE dissociate Hsp26 from Hsp26–MDH assemblies. Fractions were collected from the top of the gradient and MDH and Hsp26 were visualized by Western blot following SDS–PAGE. MDH and Hsp26 levels in each fraction were determined by quantitative immunoblot analysis using MDH‐ and Hsp26‐specific antibodies, respectively. The band intensities of Hsp26 and MDH retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and presented on the graphs on the left. Hsp26–MDH assemblies (approximate stoichiometry 1.25:1), DnaK, DnaJ, GrpE and ClpB were present at 400 nM (calculated for MDH monomers), 1 μM, 0.3 μM, 0.3 μM and 1.5 μM concentrations, respectively. Source data are available online for this figure.

Techniques Used: In Vitro, Isolation, Sedimentation, Incubation, Western Blot, SDS Page

3) Product Images from "Lack of Skeletal Muscle IL-6 Affects Pyruvate Dehydrogenase Activity at Rest and during Prolonged Exercise"

Article Title: Lack of Skeletal Muscle IL-6 Affects Pyruvate Dehydrogenase Activity at Rest and during Prolonged Exercise

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156460

Representative blots of STAT3, STAT3 Tyr705 phosphorylation (phos), AMPK Thr 172 phos, AMPKα2, ACC2 Ser212 phos, ACC2, PDH Ser293 phos, PDH Ser300 phos, PDH Ser232 phos, PDH Ser295 phos, PDH protein, lysine acetylated pdh-E1α protein, sirtuin 3 (SIRT3), Hexokinase II (HKII), GLUT4, OXPHOS complexes I-V, PDK1, PDK2, PDK4 and PDP1 protein content.
Figure Legend Snippet: Representative blots of STAT3, STAT3 Tyr705 phosphorylation (phos), AMPK Thr 172 phos, AMPKα2, ACC2 Ser212 phos, ACC2, PDH Ser293 phos, PDH Ser300 phos, PDH Ser232 phos, PDH Ser295 phos, PDH protein, lysine acetylated pdh-E1α protein, sirtuin 3 (SIRT3), Hexokinase II (HKII), GLUT4, OXPHOS complexes I-V, PDK1, PDK2, PDK4 and PDP1 protein content.

Techniques Used:

A) AMP-activated protein kinase (AMPK) Thr172 phosphorylation and B) Acetyl-CoA carboxylase 2 (ACC2) phosphorylation in skeletal muscle from skeletal muscle specific IL-6 knockout (IL-6 MKO) and littermate floxed controls (Control) mice at rest and after 10, 60 or 120 min of exercise. Values are given as mean ± SE; n = 9–10. Phosphorylation levels are given in arbitrary units (AU). *: significantly different from rest within given genotype, P
Figure Legend Snippet: A) AMP-activated protein kinase (AMPK) Thr172 phosphorylation and B) Acetyl-CoA carboxylase 2 (ACC2) phosphorylation in skeletal muscle from skeletal muscle specific IL-6 knockout (IL-6 MKO) and littermate floxed controls (Control) mice at rest and after 10, 60 or 120 min of exercise. Values are given as mean ± SE; n = 9–10. Phosphorylation levels are given in arbitrary units (AU). *: significantly different from rest within given genotype, P

Techniques Used: Knock-Out, Mouse Assay

4) Product Images from "Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]"

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]

Journal: Plant Physiology

doi: 10.1104/pp.114.236448

14-3-3 is transferred from NtCDPK1 to RSG. A, RSG and 14-3-3 dissociate from NtCDPK1 after RSG is phosphorylated by NtCDPK1. GST-NtCDPK1 was heterotrimerized with RSG and 14-3-3 as described above. The pull-down buffer was replaced with a phosphorylation
Figure Legend Snippet: 14-3-3 is transferred from NtCDPK1 to RSG. A, RSG and 14-3-3 dissociate from NtCDPK1 after RSG is phosphorylated by NtCDPK1. GST-NtCDPK1 was heterotrimerized with RSG and 14-3-3 as described above. The pull-down buffer was replaced with a phosphorylation

Techniques Used:

Model of 14-3-3 transfer from NtCDPK1 to RSG. A, In the absence of Ca 2+ , the catalytic activity of NtCDPK1 is inhibited by the autoinhibitory domain. RSG and 14-3-3 cannot access NtCDPK1. B, In the presence of Ca 2+ , the autoinhibitory domain is dissociated
Figure Legend Snippet: Model of 14-3-3 transfer from NtCDPK1 to RSG. A, In the absence of Ca 2+ , the catalytic activity of NtCDPK1 is inhibited by the autoinhibitory domain. RSG and 14-3-3 cannot access NtCDPK1. B, In the presence of Ca 2+ , the autoinhibitory domain is dissociated

Techniques Used: Activity Assay

14-3-3 shows higher affinity for RSG than for NtCDPK1. A, Autophosphorylated MBP-NtCDPK1 and phosphorylated MBP-RSG were immobilized on amylose resin and incubated with His-14-3-3. NtCDPK1- or RSG-bound proteins were subjected to SDS-PAGE, followed by
Figure Legend Snippet: 14-3-3 shows higher affinity for RSG than for NtCDPK1. A, Autophosphorylated MBP-NtCDPK1 and phosphorylated MBP-RSG were immobilized on amylose resin and incubated with His-14-3-3. NtCDPK1- or RSG-bound proteins were subjected to SDS-PAGE, followed by

Techniques Used: Incubation, SDS Page

NtCDPK1 forms a heterotrimer with RSG and 14-3-3. A, RSG binds to the complex of NtCDPK1 with 14-3-3. GST-NtCDPK1 was autophosphorylated and absorbed into glutathione beads. The immobilized GST-NtCDPK1 was incubated with His-14-3-3. Glutathione beads
Figure Legend Snippet: NtCDPK1 forms a heterotrimer with RSG and 14-3-3. A, RSG binds to the complex of NtCDPK1 with 14-3-3. GST-NtCDPK1 was autophosphorylated and absorbed into glutathione beads. The immobilized GST-NtCDPK1 was incubated with His-14-3-3. Glutathione beads

Techniques Used: Incubation

NtCDPK1 largely colocalizes with RSG and 14-3-3. Constructs expressing fluorescent protein fusion proteins were cotransfected into Arabidopsis T87 protoplasts. AGF1-CrFP was used as a control for nuclear localization. After 24 h, the cells were visualized
Figure Legend Snippet: NtCDPK1 largely colocalizes with RSG and 14-3-3. Constructs expressing fluorescent protein fusion proteins were cotransfected into Arabidopsis T87 protoplasts. AGF1-CrFP was used as a control for nuclear localization. After 24 h, the cells were visualized

Techniques Used: Construct, Expressing

The 14-3-3 binding mode of NtCDPK1 differs from that of RSG. A, The phosphorylation of Ser-114 in RSG is protected from λ-phosphatase by 14-3-3. MBP-RSG was phosphorylated by GST-NtCDPK1 with or without ATP. MBP-RSG was immobilized on amylose
Figure Legend Snippet: The 14-3-3 binding mode of NtCDPK1 differs from that of RSG. A, The phosphorylation of Ser-114 in RSG is protected from λ-phosphatase by 14-3-3. MBP-RSG was phosphorylated by GST-NtCDPK1 with or without ATP. MBP-RSG was immobilized on amylose

Techniques Used: Binding Assay

5) Product Images from "Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding"

Article Title: Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding

Journal: The EMBO Journal

doi: 10.15252/embj.201593378

KJE acts prior to the disaggregation step of IbpAB–luciferase assemblies An order of addition experiment. Luciferase (1.5 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM), diluted to 40 nM and incubated first for 20 min with the components listed in the legend and then complemented with the missing components of the functional ClpB‐KJE bichaperone system. The DnaK, DnaJ, GrpE, ClpB concentrations used were as follows: 1, 0.3, 0.3 and 1.5 μM. Data are the mean ± SD of three independent experiments.
Figure Legend Snippet: KJE acts prior to the disaggregation step of IbpAB–luciferase assemblies An order of addition experiment. Luciferase (1.5 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM), diluted to 40 nM and incubated first for 20 min with the components listed in the legend and then complemented with the missing components of the functional ClpB‐KJE bichaperone system. The DnaK, DnaJ, GrpE, ClpB concentrations used were as follows: 1, 0.3, 0.3 and 1.5 μM. Data are the mean ± SD of three independent experiments.

Techniques Used: Luciferase, Incubation, Functional Assay

Hsp70, independently of Hsp100, releases IbpAB‐dependent inhibition of disaggregation IbpAB association with GFP aggregates blocks substrate translocation through the central Hsp100 pore. Aggregated GFP (1.5 μM) was incubated for 10 min at 48°C (marked “T” in the Figure) alone (blue) or with IbpA and IbpB (3 μM and 7 μM, respectively; sky blue) or aggregated GFP and IbpAB were incubated separately at 48°C and then mixed (orange). Hsp104 D484K hyperactive variant (1 μM) was added, and GFP reactivation was monitored. KJE machinery allows to overcome the IbpAB‐dependent inhibition of IbpAB–GFP disaggregation by hyperactive Hsp104 D484K. Aggregated GFP was incubated alone at 48°C (blue and orange) or with IbpAB at 48°C (sky blue and black). After addition of the Hsp104 D484K hyperactive mutant (all plots) and KJE machinery (black and magenta), GFP reactivation was monitored. DnaK, DnaJ and GrpE were present at 1 μM, 0.3 μM and 0.3 μM. Aggregated GFP, IbpAB and Hsp104 D484K were in concentrations as in (A).
Figure Legend Snippet: Hsp70, independently of Hsp100, releases IbpAB‐dependent inhibition of disaggregation IbpAB association with GFP aggregates blocks substrate translocation through the central Hsp100 pore. Aggregated GFP (1.5 μM) was incubated for 10 min at 48°C (marked “T” in the Figure) alone (blue) or with IbpA and IbpB (3 μM and 7 μM, respectively; sky blue) or aggregated GFP and IbpAB were incubated separately at 48°C and then mixed (orange). Hsp104 D484K hyperactive variant (1 μM) was added, and GFP reactivation was monitored. KJE machinery allows to overcome the IbpAB‐dependent inhibition of IbpAB–GFP disaggregation by hyperactive Hsp104 D484K. Aggregated GFP was incubated alone at 48°C (blue and orange) or with IbpAB at 48°C (sky blue and black). After addition of the Hsp104 D484K hyperactive mutant (all plots) and KJE machinery (black and magenta), GFP reactivation was monitored. DnaK, DnaJ and GrpE were present at 1 μM, 0.3 μM and 0.3 μM. Aggregated GFP, IbpAB and Hsp104 D484K were in concentrations as in (A).

Techniques Used: Inhibition, Translocation Assay, Incubation, Variant Assay, Mutagenesis

KJE dissociates IbpAB from IbpAB–MDH assemblies in vitro Experimental scheme. IbpAB–MDH assemblies, isolated from unbound IbpAB via sedimentation in glycerol gradient, were incubated with the indicated components for 120 min and resubjected to a second round of sedimentation. KJE dissociates IbpAB from IbpAB–MDH assemblies. Fractions were collected from the top of the gradient, and MDH and IbpA were visualized by Western blot following SDS–PAGE. IbpA and MDH levels in each fraction were determined by quantitative immunoblot analysis using IbpA‐ and MDH‐specific antibodies, respectively. The band intensities of IbpA and MDH retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and plotted on the graphs on the left. IbpAB–MDH assemblies (approximate stoichiometry 1:0.5:1), DnaK, DnaJ, GrpE and ClpB were present at 400 nM (calculated for MDH monomers), 1 μM, 0.3 μM, 0.3 μM and 1.5 μM concentrations, respectively. Source data are available online for this figure.
Figure Legend Snippet: KJE dissociates IbpAB from IbpAB–MDH assemblies in vitro Experimental scheme. IbpAB–MDH assemblies, isolated from unbound IbpAB via sedimentation in glycerol gradient, were incubated with the indicated components for 120 min and resubjected to a second round of sedimentation. KJE dissociates IbpAB from IbpAB–MDH assemblies. Fractions were collected from the top of the gradient, and MDH and IbpA were visualized by Western blot following SDS–PAGE. IbpA and MDH levels in each fraction were determined by quantitative immunoblot analysis using IbpA‐ and MDH‐specific antibodies, respectively. The band intensities of IbpA and MDH retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and plotted on the graphs on the left. IbpAB–MDH assemblies (approximate stoichiometry 1:0.5:1), DnaK, DnaJ, GrpE and ClpB were present at 400 nM (calculated for MDH monomers), 1 μM, 0.3 μM, 0.3 μM and 1.5 μM concentrations, respectively. Source data are available online for this figure.

Techniques Used: In Vitro, Isolation, Sedimentation, Incubation, Western Blot, SDS Page

Hsp70 releases sHsps from the fraction of aggregated proteins in vivo Experimental scheme. BB6410 (PA1lacO‐1) dnaK, dnaJ, lacIq , Δ clpB cells were grown at 30°C in LB medium until late log phase, heat‐shocked at 46°C for 30 min, then the culture was separated into two flasks and IPTG was added at 1 mM. At indicated time points, aliquots were taken, cells were lysed and protein aggregates were isolated. The isolated fraction of aggregates was analysed by SDS–PAGE followed by staining with Coomassie brilliant blue at the indicated time points. Levels of IbpA present in aggregates at the indicated time points were determined by quantitative immunoblot analysis using IbpA‐specific antibodies. Quantification of (B) and (C). DnaK cellular levels were determined by quantitative immunoblot analysis using DnaK‐specific antibodies. Source data are available online for this figure.
Figure Legend Snippet: Hsp70 releases sHsps from the fraction of aggregated proteins in vivo Experimental scheme. BB6410 (PA1lacO‐1) dnaK, dnaJ, lacIq , Δ clpB cells were grown at 30°C in LB medium until late log phase, heat‐shocked at 46°C for 30 min, then the culture was separated into two flasks and IPTG was added at 1 mM. At indicated time points, aliquots were taken, cells were lysed and protein aggregates were isolated. The isolated fraction of aggregates was analysed by SDS–PAGE followed by staining with Coomassie brilliant blue at the indicated time points. Levels of IbpA present in aggregates at the indicated time points were determined by quantitative immunoblot analysis using IbpA‐specific antibodies. Quantification of (B) and (C). DnaK cellular levels were determined by quantitative immunoblot analysis using DnaK‐specific antibodies. Source data are available online for this figure.

Techniques Used: In Vivo, Isolation, SDS Page, Staining

KJE acts prior to disaggregation of IbpAB–MDH assemblies An order of addition experiment. MDH (2 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM) at 47°C for 30 min. IbpAB–MDH assemblies were diluted fourfold and first incubated with the components listed in the legend and then complemented with the missing components of the functional ClpB‐KJE bichaperone system. The DnaK, DnaJ, GrpE, ClpB concentrations used were as follows: 1, 0.3, 0.3 and 1.5 μM. Data are the mean ± SD of three independent experiments.
Figure Legend Snippet: KJE acts prior to disaggregation of IbpAB–MDH assemblies An order of addition experiment. MDH (2 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM) at 47°C for 30 min. IbpAB–MDH assemblies were diluted fourfold and first incubated with the components listed in the legend and then complemented with the missing components of the functional ClpB‐KJE bichaperone system. The DnaK, DnaJ, GrpE, ClpB concentrations used were as follows: 1, 0.3, 0.3 and 1.5 μM. Data are the mean ± SD of three independent experiments.

Techniques Used: Incubation, Functional Assay

IbpAB–luciferase assemblies differ in biophysical and biochemical properties from luciferase aggregates A Dynamic light scattering graphs depicting the size distribution of the following entities: native luciferase (green), IbpAB (grey), luciferase aggregates (red), IbpAB–luciferase assemblies (blue), shown as volume distribution of analysed species. Luciferase was present at 1.5 μM, IbpA at 3 μM, IbpB at 7 μM concentration. B Scheme of the disaggregation experiment. C, D Renaturation kinetics for luciferase denatured in the presence or absence of IbpAB. Luciferase (1.5 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM), diluted to 40 nM and refolded at limited (0.3 μM; C) or elevated (2.4 μM; D) KJE machinery concentrations and standard (1.5 μM) ClpB concentration. The KJE machinery concentration is given with respect to DnaK with a constant DnaK:DnaJ:GrpE ratio 1:0.3:0.3. Data are the mean ± SD of three independent experiments. E A comparison of renaturation rates for luciferase denatured in the presence or absence of IbpAB in changing KJE and constant ClpB concentrations. The KJE machinery concentration is given with respect to DnaK with a constant DnaK:DnaJ:GrpE ratio 1:0.3:0.3. F A comparison of renaturation rates for luciferase denatured in the presence or absence of IbpAB in changing ClpB and constant KJE concentrations. The dashed line indicates the theoretical scenario that luciferase refolding from aggregates and sHsp assemblies proceeds with identical refolding rates.
Figure Legend Snippet: IbpAB–luciferase assemblies differ in biophysical and biochemical properties from luciferase aggregates A Dynamic light scattering graphs depicting the size distribution of the following entities: native luciferase (green), IbpAB (grey), luciferase aggregates (red), IbpAB–luciferase assemblies (blue), shown as volume distribution of analysed species. Luciferase was present at 1.5 μM, IbpA at 3 μM, IbpB at 7 μM concentration. B Scheme of the disaggregation experiment. C, D Renaturation kinetics for luciferase denatured in the presence or absence of IbpAB. Luciferase (1.5 μM) was denatured in the presence of IbpA (3 μM) and IbpB (7 μM), diluted to 40 nM and refolded at limited (0.3 μM; C) or elevated (2.4 μM; D) KJE machinery concentrations and standard (1.5 μM) ClpB concentration. The KJE machinery concentration is given with respect to DnaK with a constant DnaK:DnaJ:GrpE ratio 1:0.3:0.3. Data are the mean ± SD of three independent experiments. E A comparison of renaturation rates for luciferase denatured in the presence or absence of IbpAB in changing KJE and constant ClpB concentrations. The KJE machinery concentration is given with respect to DnaK with a constant DnaK:DnaJ:GrpE ratio 1:0.3:0.3. F A comparison of renaturation rates for luciferase denatured in the presence or absence of IbpAB in changing ClpB and constant KJE concentrations. The dashed line indicates the theoretical scenario that luciferase refolding from aggregates and sHsp assemblies proceeds with identical refolding rates.

Techniques Used: Luciferase, Concentration Assay

Hsp70 releases sHsps from sHsp–substrate assemblies in vitro Experimental scheme. Purified IbpAB–luciferase assemblies were incubated with indicated components for the indicated time and resubjected to glycerol gradient sedimentation. KJE releases IbpAB from IbpAB–luciferase assemblies. Fractions were collected from the top of the gradient, and luciferase and IbpA were visualized by Western blot following SDS–PAGE. The band intensities of luciferase and IbpA retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and plotted on the graphs on the left. IbpA–IbpB–luciferase assemblies (approximate stoichiometry 1:0.8:1), DnaK, DnaJ and GrpE were present at 100 nM (calculated for luciferase monomer), 1 μM, 0.3 μM and 0.3 μM concentrations, respectively. Concerted action of ClpB‐KJE results in release of IbpAB and luciferase from IbpAB–luciferase assemblies. Experiments were performed and analysed as in (B). ClpB was present at 1.5 μM concentration. Source data are available online for this figure.
Figure Legend Snippet: Hsp70 releases sHsps from sHsp–substrate assemblies in vitro Experimental scheme. Purified IbpAB–luciferase assemblies were incubated with indicated components for the indicated time and resubjected to glycerol gradient sedimentation. KJE releases IbpAB from IbpAB–luciferase assemblies. Fractions were collected from the top of the gradient, and luciferase and IbpA were visualized by Western blot following SDS–PAGE. The band intensities of luciferase and IbpA retained in the core assembly (fractions 5–12) or released from the assembly (fractions 1–3) were quantified and plotted on the graphs on the left. IbpA–IbpB–luciferase assemblies (approximate stoichiometry 1:0.8:1), DnaK, DnaJ and GrpE were present at 100 nM (calculated for luciferase monomer), 1 μM, 0.3 μM and 0.3 μM concentrations, respectively. Concerted action of ClpB‐KJE results in release of IbpAB and luciferase from IbpAB–luciferase assemblies. Experiments were performed and analysed as in (B). ClpB was present at 1.5 μM concentration. Source data are available online for this figure.

Techniques Used: In Vitro, Purification, Luciferase, Incubation, Sedimentation, Western Blot, SDS Page, Concentration Assay

6) Product Images from "Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]"

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]

Journal: Plant Physiology

doi: 10.1104/pp.114.236448

The 14-3-3 binding mode of NtCDPK1 differs from that of RSG. A, The phosphorylation of Ser-114 in RSG is protected from λ-phosphatase by 14-3-3. MBP-RSG was phosphorylated by GST-NtCDPK1 with or without ATP. MBP-RSG was immobilized on amylose
Figure Legend Snippet: The 14-3-3 binding mode of NtCDPK1 differs from that of RSG. A, The phosphorylation of Ser-114 in RSG is protected from λ-phosphatase by 14-3-3. MBP-RSG was phosphorylated by GST-NtCDPK1 with or without ATP. MBP-RSG was immobilized on amylose

Techniques Used: Binding Assay

7) Product Images from "Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3"

Article Title: Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3

Journal: eLife

doi: 10.7554/eLife.05061

Silencing of all Retinal Ganglion Cells by BoTx expression leads to Ntf3 upregulation. ( A ) Expression of botulinum toxin light chain B ( Brunger et al., 2008 ; Nevin et al., 2008 ) in all RGCs leads to expansion of melanosomes similar to blind mutant fish as shown in Figure 1—figure supplement 1 . Scale bar = 200 μm. ( B ) Relative expression levels of ntf3 in 4-dpf old wildtype, kif5aa mutant, and Tg(Isl2b:Gal4, UAS:BoTxLCB-GFP) transgenic embryos. ntf3 is upregulated to a similar extent in Tg(Isl2b:Gal4, UAS:BoTxLCB-GFP) transgenic embryos as in kif5aa mutants. ( C ) Western blotting confirms the overexpression of Ntf3 protein in embryos without presynaptic activity. DOI: http://dx.doi.org/10.7554/eLife.05061.020
Figure Legend Snippet: Silencing of all Retinal Ganglion Cells by BoTx expression leads to Ntf3 upregulation. ( A ) Expression of botulinum toxin light chain B ( Brunger et al., 2008 ; Nevin et al., 2008 ) in all RGCs leads to expansion of melanosomes similar to blind mutant fish as shown in Figure 1—figure supplement 1 . Scale bar = 200 μm. ( B ) Relative expression levels of ntf3 in 4-dpf old wildtype, kif5aa mutant, and Tg(Isl2b:Gal4, UAS:BoTxLCB-GFP) transgenic embryos. ntf3 is upregulated to a similar extent in Tg(Isl2b:Gal4, UAS:BoTxLCB-GFP) transgenic embryos as in kif5aa mutants. ( C ) Western blotting confirms the overexpression of Ntf3 protein in embryos without presynaptic activity. DOI: http://dx.doi.org/10.7554/eLife.05061.020

Techniques Used: Expressing, Mutagenesis, Fluorescence In Situ Hybridization, Transgenic Assay, Western Blot, Over Expression, Activity Assay

Blumenkohl mutant RGC arbors and RGC arbors growing into a Ntf3 overexpressing tectum do not show increased filopodia dynamics. ( A ) Upper panel: Axonal arbor of a single blumenkohl mutant RGC at 5 and 7 dpf. (indicated by an arrow). D = dorsal, V = ventral, R = rostral, C = caudal. Lower panel: Tracings of an axonal arbor at time point zero. In red: Overlay of filopodia formed and retracted within 10 min (1 frame/2 min). Scale bars = 20 μm. ( B ) Upper panel: Axonal arbor of a single RGC axonal arbor growing into a Ntf3 overexpressing tectum (compare Figure 7 ) at 5 and 7 dpf. Lower panel: Tracing of an axonal arbor at time point zero. In red: Overlay of filopodia formed and retracted within 10 min (1 frame/2 min). Scale bars = 20 μm. ( C ) Quantification of filopodia numbers formed and retracted within 10 min per cell at 5 and 7 dpf. No increased rate of filopodia formation can be observed in blumenkohl mutant RGC arbors and arbors of RGCs growing into a Ntf3 overexpressing tectum while kif5aa mutant axonal arbors form and retract significantly more filopodias. Graphs for wild-type and kif5aa mutant RGC arbors are identical to Figure 7 . DOI: http://dx.doi.org/10.7554/eLife.05061.022
Figure Legend Snippet: Blumenkohl mutant RGC arbors and RGC arbors growing into a Ntf3 overexpressing tectum do not show increased filopodia dynamics. ( A ) Upper panel: Axonal arbor of a single blumenkohl mutant RGC at 5 and 7 dpf. (indicated by an arrow). D = dorsal, V = ventral, R = rostral, C = caudal. Lower panel: Tracings of an axonal arbor at time point zero. In red: Overlay of filopodia formed and retracted within 10 min (1 frame/2 min). Scale bars = 20 μm. ( B ) Upper panel: Axonal arbor of a single RGC axonal arbor growing into a Ntf3 overexpressing tectum (compare Figure 7 ) at 5 and 7 dpf. Lower panel: Tracing of an axonal arbor at time point zero. In red: Overlay of filopodia formed and retracted within 10 min (1 frame/2 min). Scale bars = 20 μm. ( C ) Quantification of filopodia numbers formed and retracted within 10 min per cell at 5 and 7 dpf. No increased rate of filopodia formation can be observed in blumenkohl mutant RGC arbors and arbors of RGCs growing into a Ntf3 overexpressing tectum while kif5aa mutant axonal arbors form and retract significantly more filopodias. Graphs for wild-type and kif5aa mutant RGC arbors are identical to Figure 7 . DOI: http://dx.doi.org/10.7554/eLife.05061.022

Techniques Used: Mutagenesis

Transplantation of kif5aa mutant RGCs into a blumenkohl mutant acceptor leads to an increased growth compared to transplantation into a wild-type acceptor. ( A ) Representative pictures of single in vivo imaged RGC axons after blastula stage transplantions from kif5aa mutant donors into a wild-type tectum (left panel) and from kif5aa mutants into a blumenkohl mutant tectum (right panel). The same cell was analyzed at 5 dpf (upper panel) and 7 dpf (middle panel). Scale bars = 20 μm. Schematics of RGC arbor complexity and size in the lower panel. In orange: Ntf3 overexpressing blumenkohl mutant tectum. D = dorsal, V = ventral, R = rostral, C = caudal. Pictures in the left panel are identical to Figure 8 . ( B ) Quantification of total branch length of transplanted RGC axons at 5 and 7 dpf. The reduced size of kif5aa mutant axonal arbors when growing into a wild-type tectum is partially rescued when transplanted into a blumenkohl mutant environment (p
Figure Legend Snippet: Transplantation of kif5aa mutant RGCs into a blumenkohl mutant acceptor leads to an increased growth compared to transplantation into a wild-type acceptor. ( A ) Representative pictures of single in vivo imaged RGC axons after blastula stage transplantions from kif5aa mutant donors into a wild-type tectum (left panel) and from kif5aa mutants into a blumenkohl mutant tectum (right panel). The same cell was analyzed at 5 dpf (upper panel) and 7 dpf (middle panel). Scale bars = 20 μm. Schematics of RGC arbor complexity and size in the lower panel. In orange: Ntf3 overexpressing blumenkohl mutant tectum. D = dorsal, V = ventral, R = rostral, C = caudal. Pictures in the left panel are identical to Figure 8 . ( B ) Quantification of total branch length of transplanted RGC axons at 5 and 7 dpf. The reduced size of kif5aa mutant axonal arbors when growing into a wild-type tectum is partially rescued when transplanted into a blumenkohl mutant environment (p

Techniques Used: Transplantation Assay, Mutagenesis, In Vivo

8) Product Images from "Plasminogen in cerebrospinal fluid originates from circulating blood"

Article Title: Plasminogen in cerebrospinal fluid originates from circulating blood

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-014-0154-y

In vivo evidence of the circulating origin of plasminogen in cerebrospinal fluid. Samples were obtained from rats injected with plasminogen labelled with Alexa Fluor 488 dye (A488-Pg). Group L2 rats were challenged with lipopolysaccharide (LPS), and group C2 was challenged with saline (see flowchart in Figure 1 ). (A) . An equal volume (10 μl) of either plasma diluted 1:50 or of cerebrospinal fluid (CSF) or urine was electrophoresed, and fluorescence (F) in the gel was directly revealed using ImageQuant TL 7.0 image analysis software (upper panel). The gel was then transferred onto a polyvinylidene fluoride membrane and detected by Western blotting with a rabbit antibody to mouse plasminogen (WB, lower panel). Representative samples are shown. (B) Micrograph showing the presence of circulating A488-Pg (indicated by arrows) in the choroid plexus of an LPS-treated rats detected by direct fluorescence microscopy. 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) indicates cell nuclei. (C) Magnified images of choroid plexus of saline- and LPS-treated rats showing the presence of circulating A488-Pg (indicated by yellow arrows) only in the LPS condition, as detected by direct fluorescence microscopy. DAPI staining (blue) indicates cell nuclei, and collagen type IV (Col IV, red) is used as a vessel marker.
Figure Legend Snippet: In vivo evidence of the circulating origin of plasminogen in cerebrospinal fluid. Samples were obtained from rats injected with plasminogen labelled with Alexa Fluor 488 dye (A488-Pg). Group L2 rats were challenged with lipopolysaccharide (LPS), and group C2 was challenged with saline (see flowchart in Figure 1 ). (A) . An equal volume (10 μl) of either plasma diluted 1:50 or of cerebrospinal fluid (CSF) or urine was electrophoresed, and fluorescence (F) in the gel was directly revealed using ImageQuant TL 7.0 image analysis software (upper panel). The gel was then transferred onto a polyvinylidene fluoride membrane and detected by Western blotting with a rabbit antibody to mouse plasminogen (WB, lower panel). Representative samples are shown. (B) Micrograph showing the presence of circulating A488-Pg (indicated by arrows) in the choroid plexus of an LPS-treated rats detected by direct fluorescence microscopy. 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) indicates cell nuclei. (C) Magnified images of choroid plexus of saline- and LPS-treated rats showing the presence of circulating A488-Pg (indicated by yellow arrows) only in the LPS condition, as detected by direct fluorescence microscopy. DAPI staining (blue) indicates cell nuclei, and collagen type IV (Col IV, red) is used as a vessel marker.

Techniques Used: In Vivo, Injection, Fluorescence, Software, Western Blot, Microscopy, Staining, Marker

9) Product Images from "Elevated O-GlcNAcylation of Extracellular Vesicle Proteins Derived from Metastatic Colorectal Cancer Cells"

Article Title: Elevated O-GlcNAcylation of Extracellular Vesicle Proteins Derived from Metastatic Colorectal Cancer Cells

Journal: Cancer Genomics & Proteomics

doi:

EVs isolated from conditioned medium of SW480 and SW620 cells containing O-GlcNAcylated proteins. A: Representative captured pictures using transmission electron microscopy revealing enriched EVs of SW480 and SW620 cells varied in size from 40-80 nm. Scale bar = 20 nm. B: Stain-free SDS-PAGE gel demonstrated the pattern of total proteins from cell lysates cultured in serum free-medium (SF-cell lysates) and different fractions of EV preparation steps (CCM, post-EVs and EVs). C: Detection of O-GlcNAcylated proteins from SF-cell lysates and different fractions of EV preparation steps by Western blotting of O-GlcNAc using RL2 antibody. Exosomal markers (Alix and TSG101) showed an enrichment of exosomes in the final fraction (EVs). D: Western blots of O-GlcNAc of EV proteins from SW620 cells treated with OGA, PNGase F and on-blot β- Elimination. Post-EVs refers to sucrose cushion fraction. 480 refers to SW480, while 620 refers to SW620.
Figure Legend Snippet: EVs isolated from conditioned medium of SW480 and SW620 cells containing O-GlcNAcylated proteins. A: Representative captured pictures using transmission electron microscopy revealing enriched EVs of SW480 and SW620 cells varied in size from 40-80 nm. Scale bar = 20 nm. B: Stain-free SDS-PAGE gel demonstrated the pattern of total proteins from cell lysates cultured in serum free-medium (SF-cell lysates) and different fractions of EV preparation steps (CCM, post-EVs and EVs). C: Detection of O-GlcNAcylated proteins from SF-cell lysates and different fractions of EV preparation steps by Western blotting of O-GlcNAc using RL2 antibody. Exosomal markers (Alix and TSG101) showed an enrichment of exosomes in the final fraction (EVs). D: Western blots of O-GlcNAc of EV proteins from SW620 cells treated with OGA, PNGase F and on-blot β- Elimination. Post-EVs refers to sucrose cushion fraction. 480 refers to SW480, while 620 refers to SW620.

Techniques Used: Isolation, Transmission Assay, Electron Microscopy, Staining, SDS Page, Cell Culture, Western Blot

10) Product Images from "Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]"

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]

Journal: Plant Physiology

doi: 10.1104/pp.114.236448

14-3-3 is transferred from NtCDPK1 to RSG. A, RSG and 14-3-3 dissociate from NtCDPK1 after RSG is phosphorylated by NtCDPK1. GST-NtCDPK1 was heterotrimerized with RSG and 14-3-3 as described above. The pull-down buffer was replaced with a phosphorylation
Figure Legend Snippet: 14-3-3 is transferred from NtCDPK1 to RSG. A, RSG and 14-3-3 dissociate from NtCDPK1 after RSG is phosphorylated by NtCDPK1. GST-NtCDPK1 was heterotrimerized with RSG and 14-3-3 as described above. The pull-down buffer was replaced with a phosphorylation

Techniques Used:

Model of 14-3-3 transfer from NtCDPK1 to RSG. A, In the absence of Ca 2+ , the catalytic activity of NtCDPK1 is inhibited by the autoinhibitory domain. RSG and 14-3-3 cannot access NtCDPK1. B, In the presence of Ca 2+ , the autoinhibitory domain is dissociated
Figure Legend Snippet: Model of 14-3-3 transfer from NtCDPK1 to RSG. A, In the absence of Ca 2+ , the catalytic activity of NtCDPK1 is inhibited by the autoinhibitory domain. RSG and 14-3-3 cannot access NtCDPK1. B, In the presence of Ca 2+ , the autoinhibitory domain is dissociated

Techniques Used: Activity Assay

14-3-3 interacts with the catalytic domain of NtCDPK1. A, Schematic diagram of NtCDPK1 constructs used in the pull-down assay. The numbers indicate the position of each amino acid residue. VD, Variable N-terminal domain; CD, catalytic domain; AID, autoinhibitory
Figure Legend Snippet: 14-3-3 interacts with the catalytic domain of NtCDPK1. A, Schematic diagram of NtCDPK1 constructs used in the pull-down assay. The numbers indicate the position of each amino acid residue. VD, Variable N-terminal domain; CD, catalytic domain; AID, autoinhibitory

Techniques Used: Construct, Pull Down Assay

14-3-3 shows higher affinity for RSG than for NtCDPK1. A, Autophosphorylated MBP-NtCDPK1 and phosphorylated MBP-RSG were immobilized on amylose resin and incubated with His-14-3-3. NtCDPK1- or RSG-bound proteins were subjected to SDS-PAGE, followed by
Figure Legend Snippet: 14-3-3 shows higher affinity for RSG than for NtCDPK1. A, Autophosphorylated MBP-NtCDPK1 and phosphorylated MBP-RSG were immobilized on amylose resin and incubated with His-14-3-3. NtCDPK1- or RSG-bound proteins were subjected to SDS-PAGE, followed by

Techniques Used: Incubation, SDS Page

14-3-3 has no effect on the kinase activity of NtCDPK1. Autophosphorylated GST-NtCDPK1 was immobilized on glutathione beads and incubated with or without His-14-3-3 in the presence of Ca 2+ . Glutathione beads into which GST-NtCDPK1 and His-14-3-3 had been
Figure Legend Snippet: 14-3-3 has no effect on the kinase activity of NtCDPK1. Autophosphorylated GST-NtCDPK1 was immobilized on glutathione beads and incubated with or without His-14-3-3 in the presence of Ca 2+ . Glutathione beads into which GST-NtCDPK1 and His-14-3-3 had been

Techniques Used: Activity Assay, Incubation

NtCDPK1 forms a heterotrimer with RSG and 14-3-3. A, RSG binds to the complex of NtCDPK1 with 14-3-3. GST-NtCDPK1 was autophosphorylated and absorbed into glutathione beads. The immobilized GST-NtCDPK1 was incubated with His-14-3-3. Glutathione beads
Figure Legend Snippet: NtCDPK1 forms a heterotrimer with RSG and 14-3-3. A, RSG binds to the complex of NtCDPK1 with 14-3-3. GST-NtCDPK1 was autophosphorylated and absorbed into glutathione beads. The immobilized GST-NtCDPK1 was incubated with His-14-3-3. Glutathione beads

Techniques Used: Incubation

NtCDPK1 largely colocalizes with RSG and 14-3-3. Constructs expressing fluorescent protein fusion proteins were cotransfected into Arabidopsis T87 protoplasts. AGF1-CrFP was used as a control for nuclear localization. After 24 h, the cells were visualized
Figure Legend Snippet: NtCDPK1 largely colocalizes with RSG and 14-3-3. Constructs expressing fluorescent protein fusion proteins were cotransfected into Arabidopsis T87 protoplasts. AGF1-CrFP was used as a control for nuclear localization. After 24 h, the cells were visualized

Techniques Used: Construct, Expressing

14-3-3 interacts with NtCDPK1. A, 14-3-3 interacts with NtCDPK1 in an autophosphorylation- and Ca 2+ -dependent manner. GST and nonphosphorylated or autophosphorylated GST-NtCDPK1 were immobilized on glutathione beads and incubated with His-14-3-3 in the
Figure Legend Snippet: 14-3-3 interacts with NtCDPK1. A, 14-3-3 interacts with NtCDPK1 in an autophosphorylation- and Ca 2+ -dependent manner. GST and nonphosphorylated or autophosphorylated GST-NtCDPK1 were immobilized on glutathione beads and incubated with His-14-3-3 in the

Techniques Used: Incubation

The 14-3-3 binding mode of NtCDPK1 differs from that of RSG. A, The phosphorylation of Ser-114 in RSG is protected from λ-phosphatase by 14-3-3. MBP-RSG was phosphorylated by GST-NtCDPK1 with or without ATP. MBP-RSG was immobilized on amylose
Figure Legend Snippet: The 14-3-3 binding mode of NtCDPK1 differs from that of RSG. A, The phosphorylation of Ser-114 in RSG is protected from λ-phosphatase by 14-3-3. MBP-RSG was phosphorylated by GST-NtCDPK1 with or without ATP. MBP-RSG was immobilized on amylose

Techniques Used: Binding Assay

11) Product Images from "Oncogenic RAS-induced CK1α drives nuclear FOXO proteolysis"

Article Title: Oncogenic RAS-induced CK1α drives nuclear FOXO proteolysis

Journal: Oncogene

doi: 10.1038/onc.2017.334

CK1α phosphorylates FOXO4 proteins specifically at serine residues 265 and 268 in vivo . ( a ) CLUSTALW protein sequence alignment of the FOXO isoforms revealed that the conserved CK1 phosphorylation motif is present in FOXO4. ( b ) NCBI HomoloGene protein sequence alignment of FOXO4 across different species indicates that the CK1α phosphorylation motif of FOXO4 is evolutionarily conserved. ( c ) FOXO4 is phosphorylated at S265 and S268 in vivo . Cells transiently expressing the indicated proteins for 48 h were assessed by IP and WB. FOXO4 IP was analyzed by SDS–PAGE /WB using the red channel of LI-COR for phosphoepitope antibodies and the green channel for FLAG-FOXO4. ( d ) Inhibition of PI3K, AKT, or CK1α markedly reduces FOXO4 S265/268 phosphorylation. Cells transiently expressing the indicated proteins for 48 h were treated with BKM120 (5 μ M ), MK2206 (5 μ M ), D4476 (5 μ M ) or PF670 (1 μ M ) for 4 h. FOXO4 IP was analyzed by SDS–PAGE /WB (LI-COR and enhanced chemiluminescence) with the indicated antibodies. ( e ) Depletion of CK1α markedly reduces FOXO4 S265/268 phosphorylation. Cells were transiently transfected with the indicated plasmids for 24 h. They were then transfected with the indicated siRNAs for an additional 48 h, followed by IP and SDS–PAGE /WB (LI-COR and enhanced chemiluminescence) analysis using the indicated antibodies. For c – e , merged LI-COR immunoblot panels represent the combined fluorescence signals from paired sets of p-FOXO4 S262 or S265/268 and FLAG-FOXO4. IgG H/C: IgG heavy chain; WCL: Whole cell lysate; n.s: non-specific bands. Similar results were observed in at least two independent experiments with duplicates.
Figure Legend Snippet: CK1α phosphorylates FOXO4 proteins specifically at serine residues 265 and 268 in vivo . ( a ) CLUSTALW protein sequence alignment of the FOXO isoforms revealed that the conserved CK1 phosphorylation motif is present in FOXO4. ( b ) NCBI HomoloGene protein sequence alignment of FOXO4 across different species indicates that the CK1α phosphorylation motif of FOXO4 is evolutionarily conserved. ( c ) FOXO4 is phosphorylated at S265 and S268 in vivo . Cells transiently expressing the indicated proteins for 48 h were assessed by IP and WB. FOXO4 IP was analyzed by SDS–PAGE /WB using the red channel of LI-COR for phosphoepitope antibodies and the green channel for FLAG-FOXO4. ( d ) Inhibition of PI3K, AKT, or CK1α markedly reduces FOXO4 S265/268 phosphorylation. Cells transiently expressing the indicated proteins for 48 h were treated with BKM120 (5 μ M ), MK2206 (5 μ M ), D4476 (5 μ M ) or PF670 (1 μ M ) for 4 h. FOXO4 IP was analyzed by SDS–PAGE /WB (LI-COR and enhanced chemiluminescence) with the indicated antibodies. ( e ) Depletion of CK1α markedly reduces FOXO4 S265/268 phosphorylation. Cells were transiently transfected with the indicated plasmids for 24 h. They were then transfected with the indicated siRNAs for an additional 48 h, followed by IP and SDS–PAGE /WB (LI-COR and enhanced chemiluminescence) analysis using the indicated antibodies. For c – e , merged LI-COR immunoblot panels represent the combined fluorescence signals from paired sets of p-FOXO4 S262 or S265/268 and FLAG-FOXO4. IgG H/C: IgG heavy chain; WCL: Whole cell lysate; n.s: non-specific bands. Similar results were observed in at least two independent experiments with duplicates.

Techniques Used: In Vivo, Sequencing, Expressing, Western Blot, SDS Page, Inhibition, Transfection, Fluorescence

12) Product Images from "Deletion of DDB1- and CUL4- associated factor-17 (Dcaf17) gene causes spermatogenesis defects and male infertility in mice"

Article Title: Deletion of DDB1- and CUL4- associated factor-17 (Dcaf17) gene causes spermatogenesis defects and male infertility in mice

Journal: Scientific Reports

doi: 10.1038/s41598-018-27379-0

Diagrammatic representation (A) of Dcaf17 gene targeting approach in mouse by homologous recombination and genotyping ( B , C ) of different alleles of Dcaf17 in mice. ( A ) Homologous recombination strategy in mouse ES cells. The Dcaf17 targeting vector (top) was constructed to replace wild type exon 4 and introduce neomycin drug selection marker, LoxP and FRT sites. ( B ) Agarose gel image of PCR genotyping of representative Dcaf17 mutant mice. PCR amplification of wild type genotype gives 1 kbps amplicon (1, C3), heterozygous genotype for Dcaf17 mutation gives 1 kbps and 193 bps amplicons (3–5, C1) and homozygous genotype for Dcaf17 mutation gives 193 bps amplicon (2, 6 and C2). 1–6 – genomic DNA samples of different Dcaf17 genotypes; C1-C3 – Different Dcaf17 genotype controls; −Ve – no template control. ( C ) Agarose gel image of RT-PCR of different Dcaf17 genotypes. PCR products of various Dcaf17 alleles and β-actin were run on the same agarose gel and single image was taken. +/+ - Dcaf17 +/+ (WT); +/− - Dcaf17 +/− (heterozygous Dcaf17 mutant); −/− - Dcaf17 −/− (homozygous Dcaf17 mutant); −Ve – no template control; M – DNA ladder. PCR fragment size for β-actin is 190 bps; for Dcaf17 +/+ is 284 bps and for Dcaf17 −/− is 148 bps. Gel images were taken using ImageQuant LAS 4000 imaging system.
Figure Legend Snippet: Diagrammatic representation (A) of Dcaf17 gene targeting approach in mouse by homologous recombination and genotyping ( B , C ) of different alleles of Dcaf17 in mice. ( A ) Homologous recombination strategy in mouse ES cells. The Dcaf17 targeting vector (top) was constructed to replace wild type exon 4 and introduce neomycin drug selection marker, LoxP and FRT sites. ( B ) Agarose gel image of PCR genotyping of representative Dcaf17 mutant mice. PCR amplification of wild type genotype gives 1 kbps amplicon (1, C3), heterozygous genotype for Dcaf17 mutation gives 1 kbps and 193 bps amplicons (3–5, C1) and homozygous genotype for Dcaf17 mutation gives 193 bps amplicon (2, 6 and C2). 1–6 – genomic DNA samples of different Dcaf17 genotypes; C1-C3 – Different Dcaf17 genotype controls; −Ve – no template control. ( C ) Agarose gel image of RT-PCR of different Dcaf17 genotypes. PCR products of various Dcaf17 alleles and β-actin were run on the same agarose gel and single image was taken. +/+ - Dcaf17 +/+ (WT); +/− - Dcaf17 +/− (heterozygous Dcaf17 mutant); −/− - Dcaf17 −/− (homozygous Dcaf17 mutant); −Ve – no template control; M – DNA ladder. PCR fragment size for β-actin is 190 bps; for Dcaf17 +/+ is 284 bps and for Dcaf17 −/− is 148 bps. Gel images were taken using ImageQuant LAS 4000 imaging system.

Techniques Used: Homologous Recombination, Mouse Assay, Plasmid Preparation, Construct, Introduce, Selection, Marker, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Mutagenesis, Amplification, Reverse Transcription Polymerase Chain Reaction, Imaging

13) Product Images from "Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]"

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]

Journal: Plant Physiology

doi: 10.1104/pp.114.236448

14-3-3 is transferred from NtCDPK1 to RSG. A, RSG and 14-3-3 dissociate from NtCDPK1 after RSG is phosphorylated by NtCDPK1. GST-NtCDPK1 was heterotrimerized with RSG and 14-3-3 as described above. The pull-down buffer was replaced with a phosphorylation
Figure Legend Snippet: 14-3-3 is transferred from NtCDPK1 to RSG. A, RSG and 14-3-3 dissociate from NtCDPK1 after RSG is phosphorylated by NtCDPK1. GST-NtCDPK1 was heterotrimerized with RSG and 14-3-3 as described above. The pull-down buffer was replaced with a phosphorylation

Techniques Used:

14-3-3 has no effect on the kinase activity of NtCDPK1. Autophosphorylated GST-NtCDPK1 was immobilized on glutathione beads and incubated with or without His-14-3-3 in the presence of Ca 2+ . Glutathione beads into which GST-NtCDPK1 and His-14-3-3 had been
Figure Legend Snippet: 14-3-3 has no effect on the kinase activity of NtCDPK1. Autophosphorylated GST-NtCDPK1 was immobilized on glutathione beads and incubated with or without His-14-3-3 in the presence of Ca 2+ . Glutathione beads into which GST-NtCDPK1 and His-14-3-3 had been

Techniques Used: Activity Assay, Incubation

NtCDPK1 forms a heterotrimer with RSG and 14-3-3. A, RSG binds to the complex of NtCDPK1 with 14-3-3. GST-NtCDPK1 was autophosphorylated and absorbed into glutathione beads. The immobilized GST-NtCDPK1 was incubated with His-14-3-3. Glutathione beads
Figure Legend Snippet: NtCDPK1 forms a heterotrimer with RSG and 14-3-3. A, RSG binds to the complex of NtCDPK1 with 14-3-3. GST-NtCDPK1 was autophosphorylated and absorbed into glutathione beads. The immobilized GST-NtCDPK1 was incubated with His-14-3-3. Glutathione beads

Techniques Used: Incubation

14-3-3 interacts with NtCDPK1. A, 14-3-3 interacts with NtCDPK1 in an autophosphorylation- and Ca 2+ -dependent manner. GST and nonphosphorylated or autophosphorylated GST-NtCDPK1 were immobilized on glutathione beads and incubated with His-14-3-3 in the
Figure Legend Snippet: 14-3-3 interacts with NtCDPK1. A, 14-3-3 interacts with NtCDPK1 in an autophosphorylation- and Ca 2+ -dependent manner. GST and nonphosphorylated or autophosphorylated GST-NtCDPK1 were immobilized on glutathione beads and incubated with His-14-3-3 in the

Techniques Used: Incubation

The 14-3-3 binding mode of NtCDPK1 differs from that of RSG. A, The phosphorylation of Ser-114 in RSG is protected from λ-phosphatase by 14-3-3. MBP-RSG was phosphorylated by GST-NtCDPK1 with or without ATP. MBP-RSG was immobilized on amylose
Figure Legend Snippet: The 14-3-3 binding mode of NtCDPK1 differs from that of RSG. A, The phosphorylation of Ser-114 in RSG is protected from λ-phosphatase by 14-3-3. MBP-RSG was phosphorylated by GST-NtCDPK1 with or without ATP. MBP-RSG was immobilized on amylose

Techniques Used: Binding Assay

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Immunohistochemistry:

Article Title: LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors
Article Snippet: .. For autophagy studies we used LC3 (1:500 immunoblots) from Novus and p62 (1:500 for immunoblots and 1:2000 for immunohistochemistry) from Progen. .. Neurogenesis studies utilized rat α-5-bromo-2-deoxyuridine (BrdU) 1:500 (Oxford Biotechnology) and goat α-doublecortin (DCX) 1:500, (Santa Cruz Biotechnology).

Incubation:

Article Title: HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress
Article Snippet: .. Membranes were incubated overnight at 4 °C with antibodies reactive to the following proteins: ubiquitin (P4D1) (Cell Signaling, 3936, 1:1,000), p62 (Progen, GP62C, 1:1,000), LC3 (Novus, NB100-2331, 1:500), GFP (Life Technologies, A11121), HA (Monoclonal Antibody Facility, Hospital for Sick Children, 12CA5, 1:1,000), Unc-45b (GenWay, EV0087,1:1,000), gelsolin (BD, 610412, 1:1,000), vimentin (R28) (Cell Signaling, 3932, 1:1,000), Fhl1 (Novus, NB100-1461, 1:1,000) vinculin (Sigma, V4505, 1:1,000), β-tubulin (Sigma, T8328, 1:1,000), caldesmon (Abcam, ab32330, 1:1,000), Hsc70 (Abcam, ab2788, 1:1,000), Hsp70 (Stressgen, SPA810, 1:1,000), Hsp25 (Stressgen, SPA801, 1:1,000), connexin 40 (Life Technologies, 36-5000, 1:1,000) and connexin 43 (Life Technologies, 71-0700, 1:1,000). .. Blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad, 170-5046, 1:50,000), goat anti-rabbit IgG (Bio-Rad, 170-5047, 1:100,000), monoclonal mouse anti-goat IgG (Jackson ImmunoResearch, 205-032-176, 1:50,000) or goat anti-guinea pig IgG (Jackson ImmunoResearch, 106-035-003, 1:100,000) and developed using ECL or ECL Plus Western Blotting Detection System (Amersham Biosciences).

other:

Article Title: Identification of 7-(4′-Cyanophenyl)indoline-1-benzenesulfonamide as a mitotic inhibitor to induce apoptotic cell death and inhibit autophagy in human colorectal cancer cells
Article Snippet: LC3 was purchased from Novus (Littleton, CO, USA).

Staining:

Article Title: Environmental Enrichment Rescues Protein Deficits in a Mouse Model of Huntington's Disease, Indicating a Possible Disease Mechanism
Article Snippet: .. After amido staining, membranes were rinsed, then probed for BDNF, NGF, or DARPP-32 immunoreactivity using ECL (Amersham, Little Chalfont, UK) according to the manufacturer's instructions. .. Briefly, membranes were blocked for 1 hr in 5% powdered milk in PBS buffer containing 0.1% Triton detergent (PBST; Sigma) at pH 7.4, followed by a 2 hr incubation in primary antibodies to BDNF (Santa Cruz 546; Santa Cruz Biotechnology, Santa Cruz, CA), NGF (Santa Cruz Biotechnology), or DARPP-32 (Chemicon, Temecula, CA) diluted in PBST to final concentrations of 1:1000 BDNF, 1:1000 NGF, or 1:10,000 DARPP-32.

Article Title: Defective i6A37 Modification of Mitochondrial and Cytosolic tRNAs Results from Pathogenic Mutations in TRIT1 and Its Substrate tRNA
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Western Blot:

Article Title: LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors
Article Snippet: .. For autophagy studies we used LC3 (1:500 immunoblots) from Novus and p62 (1:500 for immunoblots and 1:2000 for immunohistochemistry) from Progen. .. Neurogenesis studies utilized rat α-5-bromo-2-deoxyuridine (BrdU) 1:500 (Oxford Biotechnology) and goat α-doublecortin (DCX) 1:500, (Santa Cruz Biotechnology).

Software:

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