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    Structured Review

    Millipore immunoblotting
    Melatonin reduces autophagy turnover thereby increasing BeWo cell vulnerability to H/R. BeWo cells were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ). (A) Sequestosome-1, SQSTM1/P62, protein expression was detected by <t>immunoblotting</t> using a polyclonal antibody in BeWo cells exposed to H/R. Cells were treated with dimethyl sulfoxide (DMSO) 0.1% or 1 mM melatonin (MEL) for 24 h. Cells were also treated with BAF A1 (10 nM) during the last 2 h of BeWo cell culture. (B) Nrf2 protein expression was detected by immunoblotting using a polyclonal antibody in BeWo cells exposed to normoxia or H/R. Sulforaphane 10 μM was added to BeWo cells for 24 h as an Nrf2 inducer. (C) Relative optical density of the carboxy-terminal catalytic domain of the poly (ADP-ribose) polymerase (PARP), which was cleaved between the Asp214 and Gly215 was normalized with total PARP. Both cleaved PARP and PARP1 were detected by immunoblotting using a polyclonal antibody. (D) The nuclear and the cytosolic contents of the Nrf2 protein of BeWo cells exposed to H/R were detected by immunoblotting. Histone H3 and α-tubulin were used as protein markers of the purity of nuclear and cytosolic fractions, respectively. Representative blots are shown below the graphs and the relative molecular mass (kDa) is indicated to the right of the blot. Total protein amounts were measured by staining with MemCode reversible protein stain. Data shown are mean ±SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (A, B, C) or Student’s t -test (D) (* p
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    Images

    1) Product Images from "Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells"

    Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202458

    Melatonin reduces autophagy turnover thereby increasing BeWo cell vulnerability to H/R. BeWo cells were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ). (A) Sequestosome-1, SQSTM1/P62, protein expression was detected by immunoblotting using a polyclonal antibody in BeWo cells exposed to H/R. Cells were treated with dimethyl sulfoxide (DMSO) 0.1% or 1 mM melatonin (MEL) for 24 h. Cells were also treated with BAF A1 (10 nM) during the last 2 h of BeWo cell culture. (B) Nrf2 protein expression was detected by immunoblotting using a polyclonal antibody in BeWo cells exposed to normoxia or H/R. Sulforaphane 10 μM was added to BeWo cells for 24 h as an Nrf2 inducer. (C) Relative optical density of the carboxy-terminal catalytic domain of the poly (ADP-ribose) polymerase (PARP), which was cleaved between the Asp214 and Gly215 was normalized with total PARP. Both cleaved PARP and PARP1 were detected by immunoblotting using a polyclonal antibody. (D) The nuclear and the cytosolic contents of the Nrf2 protein of BeWo cells exposed to H/R were detected by immunoblotting. Histone H3 and α-tubulin were used as protein markers of the purity of nuclear and cytosolic fractions, respectively. Representative blots are shown below the graphs and the relative molecular mass (kDa) is indicated to the right of the blot. Total protein amounts were measured by staining with MemCode reversible protein stain. Data shown are mean ±SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (A, B, C) or Student’s t -test (D) (* p
    Figure Legend Snippet: Melatonin reduces autophagy turnover thereby increasing BeWo cell vulnerability to H/R. BeWo cells were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ). (A) Sequestosome-1, SQSTM1/P62, protein expression was detected by immunoblotting using a polyclonal antibody in BeWo cells exposed to H/R. Cells were treated with dimethyl sulfoxide (DMSO) 0.1% or 1 mM melatonin (MEL) for 24 h. Cells were also treated with BAF A1 (10 nM) during the last 2 h of BeWo cell culture. (B) Nrf2 protein expression was detected by immunoblotting using a polyclonal antibody in BeWo cells exposed to normoxia or H/R. Sulforaphane 10 μM was added to BeWo cells for 24 h as an Nrf2 inducer. (C) Relative optical density of the carboxy-terminal catalytic domain of the poly (ADP-ribose) polymerase (PARP), which was cleaved between the Asp214 and Gly215 was normalized with total PARP. Both cleaved PARP and PARP1 were detected by immunoblotting using a polyclonal antibody. (D) The nuclear and the cytosolic contents of the Nrf2 protein of BeWo cells exposed to H/R were detected by immunoblotting. Histone H3 and α-tubulin were used as protein markers of the purity of nuclear and cytosolic fractions, respectively. Representative blots are shown below the graphs and the relative molecular mass (kDa) is indicated to the right of the blot. Total protein amounts were measured by staining with MemCode reversible protein stain. Data shown are mean ±SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (A, B, C) or Student’s t -test (D) (* p

    Techniques Used: Expressing, Cell Culture, Staining

    Melatonin and hypoxia/reoxygenation decrease the mitochondrial content in BeWo cells. BeWo cells were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ) with or without treatment with melatonin (1 mM) or bafilomycin (BAF A1: 10 nM). (A) TOMM20 protein level was detected by immunoblotting using a polyclonal antibody. The relative molecular mass (kDa) is indicated to the right of the representative blot. Total protein amounts were measured by staining with MemCode reversible protein stain. Data shown are mean ± SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (* p
    Figure Legend Snippet: Melatonin and hypoxia/reoxygenation decrease the mitochondrial content in BeWo cells. BeWo cells were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ) with or without treatment with melatonin (1 mM) or bafilomycin (BAF A1: 10 nM). (A) TOMM20 protein level was detected by immunoblotting using a polyclonal antibody. The relative molecular mass (kDa) is indicated to the right of the representative blot. Total protein amounts were measured by staining with MemCode reversible protein stain. Data shown are mean ± SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (* p

    Techniques Used: Staining

    Hypoxia/reoxygenation and melatonin differentially regulate autophagosome formation in BeWo cells. (A) BeWo cells were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ) with or without treatment with melatonin (1 mM) or rapamycin (300 nM). Protein levels of autophagy-related (ATG) ATG5-ATG12, ATG16β, ATG16α, and ATG7 were detected by immunoblotting using monoclonal antibodies. Microtubule-associated proteins 1A/1B lightchain 3B (LC3B) was detected by immunoblotting using a polyclonal antibody. The relative molecular mass (kDa) is indicated to the right of the representative blot. Total protein amounts were measured by staining with MemCode reversible protein stain. ATG16β and ATG16α were represented with two distinct blot images, separated by a dotted line. (B) Relative optical density of ATG7 normalized with total protein is expressed in comparison to vehicle control (DMSO) in normoxia. (C) Relative optical density of lipidated LC3B (LC3B-II) were normalized with total protein being expressed in comparison to vehicle control (DMSO) in normoxia. Data shown are mean ± SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (* p
    Figure Legend Snippet: Hypoxia/reoxygenation and melatonin differentially regulate autophagosome formation in BeWo cells. (A) BeWo cells were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ) with or without treatment with melatonin (1 mM) or rapamycin (300 nM). Protein levels of autophagy-related (ATG) ATG5-ATG12, ATG16β, ATG16α, and ATG7 were detected by immunoblotting using monoclonal antibodies. Microtubule-associated proteins 1A/1B lightchain 3B (LC3B) was detected by immunoblotting using a polyclonal antibody. The relative molecular mass (kDa) is indicated to the right of the representative blot. Total protein amounts were measured by staining with MemCode reversible protein stain. ATG16β and ATG16α were represented with two distinct blot images, separated by a dotted line. (B) Relative optical density of ATG7 normalized with total protein is expressed in comparison to vehicle control (DMSO) in normoxia. (C) Relative optical density of lipidated LC3B (LC3B-II) were normalized with total protein being expressed in comparison to vehicle control (DMSO) in normoxia. Data shown are mean ± SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (* p

    Techniques Used: Staining

    Melatonin increases autophagy turnover and Nrf2 expression in primary villous cytotrophoblasts exposed to hypoxia/reoxygenation. Human primary villous cytotrophoblasts (vCTBs) were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ). Cells were treated with dimethyl sulfoxide (DMSO) 0.1% or 1 mM melatonin (MEL) for 24 h. (A) Protein expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Beclin-1, and microtubule-associated proteins 1A/1B lightchain 3B (LC3B) were detected by immunoblotting. (B) Sequestosome-1 is also known as the ubiquitin-binding protein p62 (SQSTM1/P62), and its protein expression was detected by immunoblotting using a polyclonal antibody in vCTB exposed to H/R. Cells were treated with dimethyl sulfoxide (DMSO) 0.1% or 1 mM melatonin (MEL) for 24 h. vCTB were also treated with bafilomycin A1 (10 nM) during the last 2 h of culture. Total protein amounts were measured by staining with MemCode reversible protein stain. The relative molecular mass (kDa) is indicated to the right of the representative blot. (C) Relative optical density of Nrf2 normalized with total protein. Data shown are mean ± SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (* p
    Figure Legend Snippet: Melatonin increases autophagy turnover and Nrf2 expression in primary villous cytotrophoblasts exposed to hypoxia/reoxygenation. Human primary villous cytotrophoblasts (vCTBs) were exposed to normoxia (8% O 2 ; 5% CO 2 ; 87% N 2 ) or hypoxia (4 h) /reoxygenation (20 h) (H/R) (0.5% O 2 ; 5% CO 2 ; 94.5% N 2 ). Cells were treated with dimethyl sulfoxide (DMSO) 0.1% or 1 mM melatonin (MEL) for 24 h. (A) Protein expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Beclin-1, and microtubule-associated proteins 1A/1B lightchain 3B (LC3B) were detected by immunoblotting. (B) Sequestosome-1 is also known as the ubiquitin-binding protein p62 (SQSTM1/P62), and its protein expression was detected by immunoblotting using a polyclonal antibody in vCTB exposed to H/R. Cells were treated with dimethyl sulfoxide (DMSO) 0.1% or 1 mM melatonin (MEL) for 24 h. vCTB were also treated with bafilomycin A1 (10 nM) during the last 2 h of culture. Total protein amounts were measured by staining with MemCode reversible protein stain. The relative molecular mass (kDa) is indicated to the right of the representative blot. (C) Relative optical density of Nrf2 normalized with total protein. Data shown are mean ± SD and were analyzed using ANOVA, followed by Student-Newman-Keuls (* p

    Techniques Used: Expressing, Derivative Assay, Binding Assay, Staining

    2) Product Images from "A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *"

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.750570

    Splicing factors are part of high molecular weight complexes. Crude cell extracts of R4T, RT2T, R6T, and R2T were subjected to size exclusion chromatography, and 18 fractions were analyzed by immunoblotting with antiserum against the TAP sequence. As a control, the large subunit of Rubisco, which has an estimated size of 560 kDa, was detected using an α-rbcL antibody. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.
    Figure Legend Snippet: Splicing factors are part of high molecular weight complexes. Crude cell extracts of R4T, RT2T, R6T, and R2T were subjected to size exclusion chromatography, and 18 fractions were analyzed by immunoblotting with antiserum against the TAP sequence. As a control, the large subunit of Rubisco, which has an estimated size of 560 kDa, was detected using an α-rbcL antibody. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography, Sequencing, Filtration

    3) Product Images from "MP470, a novel receptor tyrosine kinase inhibitor, in combination with Erlotinib inhibits the HER family/PI3K/Akt pathway and tumor growth in prostate cancer"

    Article Title: MP470, a novel receptor tyrosine kinase inhibitor, in combination with Erlotinib inhibits the HER family/PI3K/Akt pathway and tumor growth in prostate cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-142

    Effects of MP470, Erlotinib, and MP470-Erlotinib treatment on Akt activity . (a). LNCaP cells were treated with DMSO or different doses of Erlotinib, MP470, IM or combinations as indicated for 30 hr. Phospho(Ser473)-Akt and total Akt were detected by immunoblotting. β-actin antibody was used as the loading control. (b). LNCaP cells were grown in androgen-depleted medium, phenol red-free RPMI 1640 supplemented with 10% charcoal/dextran-treated FBS for 10 days. The cells were treated with 10 μM of Erlotinib, MP470, IM alone or Erlotinib plus MP470 and Erlotinib plus IM for 24 hr, and Akt phosphorylation was analyzed by Western blotting.
    Figure Legend Snippet: Effects of MP470, Erlotinib, and MP470-Erlotinib treatment on Akt activity . (a). LNCaP cells were treated with DMSO or different doses of Erlotinib, MP470, IM or combinations as indicated for 30 hr. Phospho(Ser473)-Akt and total Akt were detected by immunoblotting. β-actin antibody was used as the loading control. (b). LNCaP cells were grown in androgen-depleted medium, phenol red-free RPMI 1640 supplemented with 10% charcoal/dextran-treated FBS for 10 days. The cells were treated with 10 μM of Erlotinib, MP470, IM alone or Erlotinib plus MP470 and Erlotinib plus IM for 24 hr, and Akt phosphorylation was analyzed by Western blotting.

    Techniques Used: Activity Assay, Western Blot

    MP470-Erlotinib combination markedly reduces protein kinase activity . (a-b). LNCaP (a) and NIH3T3 (b) cells were serum starved for 24 hr, pretreated with drugs as indicated for 2 hr, and then treated with pervanadate (100 μM) for 10 min. Whole cell extracts were analyzed by immunoblotting for phosphorylated tyrosine kinases, phosphorylated Akt (Ser473), phosphorylated ERK1/2 (Thr202/Tyr204), and total Akt. (c). LNCaP cells were serum-starved for 24 hr, pretreated with DMSO, 10 μM of MP470 or MP470-Erlotinib, and then stimulated by pervanadate for 10 min. For immunoprecipitation (IP) assays, whole cell extracts (WCE) containing equal amounts of protein were incubated with anti-phosphotyrosine (PY20) antibodies overnight at 4°C. Immune complexes were enriched by Protein G-Agarose beads and probed by Western blotting for the p85 subunit of PI3K.
    Figure Legend Snippet: MP470-Erlotinib combination markedly reduces protein kinase activity . (a-b). LNCaP (a) and NIH3T3 (b) cells were serum starved for 24 hr, pretreated with drugs as indicated for 2 hr, and then treated with pervanadate (100 μM) for 10 min. Whole cell extracts were analyzed by immunoblotting for phosphorylated tyrosine kinases, phosphorylated Akt (Ser473), phosphorylated ERK1/2 (Thr202/Tyr204), and total Akt. (c). LNCaP cells were serum-starved for 24 hr, pretreated with DMSO, 10 μM of MP470 or MP470-Erlotinib, and then stimulated by pervanadate for 10 min. For immunoprecipitation (IP) assays, whole cell extracts (WCE) containing equal amounts of protein were incubated with anti-phosphotyrosine (PY20) antibodies overnight at 4°C. Immune complexes were enriched by Protein G-Agarose beads and probed by Western blotting for the p85 subunit of PI3K.

    Techniques Used: Activity Assay, Immunoprecipitation, Incubation, Western Blot

    Effects of MP470-Erlotinib combination on tyrosine phosphorylation of the HER family and PI3K/Akt . LNCaP (a) and T47D (b) cells were serum starved for 24 hr, pretreated with 10 μM drug as indicated for 2 hr, and then treated with pervanadate (100 μM) for 10 min. Cell extracts were incubated with anti-EGFR, anti-HER2 and anti-HER3 antibodies at 4°C overnight. The immune complexes were enriched by Protein G-Agarose beads and probed by immunoblotting for phosphotyrosine (PY20) and the p85 regularly subunit of PI3K. Western blotting analysis for phosphorylated Akt was performed in T47D cells. (c). SiRNA knockdown of HER2 decreased phosphorylated Akt. LNCaP cells were grown to 70% confluence and treated with non-targeting siRNA (control RNAi) and siRNA against HER2 at a concentration of 100 nmol/L. At 72 hr, cells were harvested to detect HER2, phosphorylated Akt and total Akt by Western blotting. GAPDH was used as a loading control.
    Figure Legend Snippet: Effects of MP470-Erlotinib combination on tyrosine phosphorylation of the HER family and PI3K/Akt . LNCaP (a) and T47D (b) cells were serum starved for 24 hr, pretreated with 10 μM drug as indicated for 2 hr, and then treated with pervanadate (100 μM) for 10 min. Cell extracts were incubated with anti-EGFR, anti-HER2 and anti-HER3 antibodies at 4°C overnight. The immune complexes were enriched by Protein G-Agarose beads and probed by immunoblotting for phosphotyrosine (PY20) and the p85 regularly subunit of PI3K. Western blotting analysis for phosphorylated Akt was performed in T47D cells. (c). SiRNA knockdown of HER2 decreased phosphorylated Akt. LNCaP cells were grown to 70% confluence and treated with non-targeting siRNA (control RNAi) and siRNA against HER2 at a concentration of 100 nmol/L. At 72 hr, cells were harvested to detect HER2, phosphorylated Akt and total Akt by Western blotting. GAPDH was used as a loading control.

    Techniques Used: Incubation, Western Blot, Concentration Assay

    Effects of MP470 or the MP470-Erlotinib combination on prostate cancer cell proliferation and apoptosis . (a). LNCaP, DU145 and PC-3 cells were exposed to varying concentrations of MP470 for 4 days. Cell viability was assessed by MTS analysis. Points are the means of triplicate determinations ± SD. The IC 50 for LNCaP and PC-3 is ~4 μM and 8 μM, respectively. Chemical structure of MP470 was shown at bottom. (b). LNCaP cells were exposed to varying concentrations of Erlotinib (Tarceva), MP470 or Erlotinib (10 μM) plus MP470 (varied concentrations) for 4 days. Cell viability was assessed by MTS analysis. IC 50 was calculated with CalcuSyn software and the graph was shown. (c). LNCaP cells were treated with the indicated doses of Erlotinib or MP470 alone or in combination for 48 hr, and apoptosis was detected as morphologic change by fluorescent microscopy. Values are the means of three independent experiments ± SD. Control cells were treated with DMSO vehicle. (d). LNCaP cells were treated with DMSO (control), 10 μM of Erlotinib or MP470 or in combination for 48 h. Flow cytometric analysis of apoptotic fraction based on propidium iodide (Y-axis) and annexin V staining (X-axis) showed up to 28% and 46% of apoptosis was induced by MP470 alone and in combination with Erlotinib, respectively. (e). LNCaP cells were treated with the indicated doses of Erlotinib or MP470 or IM alone or the combinations for 48 hr, and PARP cleavage was determined by immunoblotting with anti-PARP antibody. β-actin was used as a loading control. MP470 or MP470-Erlotinib but not Erlotinib or IM or Erlotinib-IM combination was shown to cause PARP cleavage in LNCaP cells.
    Figure Legend Snippet: Effects of MP470 or the MP470-Erlotinib combination on prostate cancer cell proliferation and apoptosis . (a). LNCaP, DU145 and PC-3 cells were exposed to varying concentrations of MP470 for 4 days. Cell viability was assessed by MTS analysis. Points are the means of triplicate determinations ± SD. The IC 50 for LNCaP and PC-3 is ~4 μM and 8 μM, respectively. Chemical structure of MP470 was shown at bottom. (b). LNCaP cells were exposed to varying concentrations of Erlotinib (Tarceva), MP470 or Erlotinib (10 μM) plus MP470 (varied concentrations) for 4 days. Cell viability was assessed by MTS analysis. IC 50 was calculated with CalcuSyn software and the graph was shown. (c). LNCaP cells were treated with the indicated doses of Erlotinib or MP470 alone or in combination for 48 hr, and apoptosis was detected as morphologic change by fluorescent microscopy. Values are the means of three independent experiments ± SD. Control cells were treated with DMSO vehicle. (d). LNCaP cells were treated with DMSO (control), 10 μM of Erlotinib or MP470 or in combination for 48 h. Flow cytometric analysis of apoptotic fraction based on propidium iodide (Y-axis) and annexin V staining (X-axis) showed up to 28% and 46% of apoptosis was induced by MP470 alone and in combination with Erlotinib, respectively. (e). LNCaP cells were treated with the indicated doses of Erlotinib or MP470 or IM alone or the combinations for 48 hr, and PARP cleavage was determined by immunoblotting with anti-PARP antibody. β-actin was used as a loading control. MP470 or MP470-Erlotinib but not Erlotinib or IM or Erlotinib-IM combination was shown to cause PARP cleavage in LNCaP cells.

    Techniques Used: Software, Microscopy, Flow Cytometry, Staining

    4) Product Images from "Repetitive N-WASP-Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly"

    Article Title: Repetitive N-WASP-Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000191

    Multiple EspF U repeats are required for efficient binding of N-WASP in brain extract. (A) N-terminally His10-tagged and C-terminally 5myc-tagged EspF U derivatives were expressed in E. coli , purified, resolved by SDS-PAGE, and stained with Coomassie blue. (B) Cobalt-chelated magnetic particles were coated with saturating concentrations of EspF U derivatives and subsequently incubated with porcine brain extract. The association of native N-WASP with EspF U -coated beads was assessed by SDS-PAGE followed by immunoblotting of bead eluates with antibodies to N-WASP and staining EspF U with Ponceau S. (C) His-EspF U -myc constructs were added to brain extract at the indicated concentrations and collected using cobalt-chelated magnetic particles. The association of native N-WASP and EspF U with the beads was assessed by immunoblotting of bead eluates with antibodies to N-WASP and staining EspF U with Ponceau S.
    Figure Legend Snippet: Multiple EspF U repeats are required for efficient binding of N-WASP in brain extract. (A) N-terminally His10-tagged and C-terminally 5myc-tagged EspF U derivatives were expressed in E. coli , purified, resolved by SDS-PAGE, and stained with Coomassie blue. (B) Cobalt-chelated magnetic particles were coated with saturating concentrations of EspF U derivatives and subsequently incubated with porcine brain extract. The association of native N-WASP with EspF U -coated beads was assessed by SDS-PAGE followed by immunoblotting of bead eluates with antibodies to N-WASP and staining EspF U with Ponceau S. (C) His-EspF U -myc constructs were added to brain extract at the indicated concentrations and collected using cobalt-chelated magnetic particles. The association of native N-WASP and EspF U with the beads was assessed by immunoblotting of bead eluates with antibodies to N-WASP and staining EspF U with Ponceau S.

    Techniques Used: Binding Assay, Purification, SDS Page, Staining, Incubation, Construct

    5) Product Images from "EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity"

    Article Title: EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004389

    EDR1 interacts with MKK4 and MKK5. ( A ) EDR1 full length (F), EDR1 N-terminal domain (N) and EDR1 C-terminal domain (C) were fused to the Gal4 DNA binding domain (BD). MKK1 , MKK2 , MKK4 , MKK5 , MPK3 and MPK6 were fused to the Gal4 transactivation domain (AD). Different pairs of constructs were cotransformed into yeast isolate AH109 to test the interaction. 10 µL suspension (OD 600 = 0.5) of each cotransformant was dropped on the synthetic dropout (SD) medium lacking Leu and Trp (left) and SD medium lacking Ade, His, Leu and Trp (right), respectively. Pictures were taken after 2 days incubation. ( B ) YFP YN -fused EDR1 and YFP YC -fused MKK4 / MKK5 were co-expressed in N . benthamiana . YFP fluorescence was detected by confocal microscopy. Cotransformants of YFP YN -EDR1 and YFP YC , YFP YN and YFP YC -MKK4, or YFP YN and YFP YC -MKK5 were used as controls. Bar = 50 µm. ( C ) EDR1 N-terminal domain was expressed alone or co-expressed with MKK4 and MKK5 in N . benthamiana . Proteins were extracted after 48 h, and subjected to immunoprecipitation by anti-HA antibody, followed by immunoblotting using anti-Myc and anti-HA antibodies, respectively. ( D ) EDR1-Flag transgenic plants and EDR1-Flag / HA-MKK5 double transgenic plants were used for co-IP. The proteins were analyzed by immunoblotting using anti-Flag or anti-HA antibody, respectively. The above experiments were repeated three times with similar results.
    Figure Legend Snippet: EDR1 interacts with MKK4 and MKK5. ( A ) EDR1 full length (F), EDR1 N-terminal domain (N) and EDR1 C-terminal domain (C) were fused to the Gal4 DNA binding domain (BD). MKK1 , MKK2 , MKK4 , MKK5 , MPK3 and MPK6 were fused to the Gal4 transactivation domain (AD). Different pairs of constructs were cotransformed into yeast isolate AH109 to test the interaction. 10 µL suspension (OD 600 = 0.5) of each cotransformant was dropped on the synthetic dropout (SD) medium lacking Leu and Trp (left) and SD medium lacking Ade, His, Leu and Trp (right), respectively. Pictures were taken after 2 days incubation. ( B ) YFP YN -fused EDR1 and YFP YC -fused MKK4 / MKK5 were co-expressed in N . benthamiana . YFP fluorescence was detected by confocal microscopy. Cotransformants of YFP YN -EDR1 and YFP YC , YFP YN and YFP YC -MKK4, or YFP YN and YFP YC -MKK5 were used as controls. Bar = 50 µm. ( C ) EDR1 N-terminal domain was expressed alone or co-expressed with MKK4 and MKK5 in N . benthamiana . Proteins were extracted after 48 h, and subjected to immunoprecipitation by anti-HA antibody, followed by immunoblotting using anti-Myc and anti-HA antibodies, respectively. ( D ) EDR1-Flag transgenic plants and EDR1-Flag / HA-MKK5 double transgenic plants were used for co-IP. The proteins were analyzed by immunoblotting using anti-Flag or anti-HA antibody, respectively. The above experiments were repeated three times with similar results.

    Techniques Used: Binding Assay, Construct, Incubation, Fluorescence, Confocal Microscopy, Immunoprecipitation, Transgenic Assay, Co-Immunoprecipitation Assay

    6) Product Images from "Inflammation and Hyperglycemia Mediate Deaf1 Splicing in the Pancreatic Lymph Nodes via Distinct Pathways During Type 1 Diabetes"

    Article Title: Inflammation and Hyperglycemia Mediate Deaf1 Splicing in the Pancreatic Lymph Nodes via Distinct Pathways During Type 1 Diabetes

    Journal: Diabetes

    doi: 10.2337/db14-0803

    PTBP2 and SRSF10 mediate the splicing of human DEAF1 . A–F : QPCR and immunoblotting data showing transfection of HEK293T cells with plasmids expressing PTBP2 or SRSF10 , alone (1 μg each) or in combination (0.5 μg each). Transfection resulted in significantly increased mRNA ( A and B ) and protein ( C ) expression of the corresponding genes 24 and 48 h after transfection. C : The antibodies used detected multiple alternatively spliced isoforms of PTBP2 and SRSF10. The ∼57-kDa product detected by the anti-PTBP2 antibody may represent the larger PTBP2 isoforms 1, 2, 3, and/or 4 (predicted size ∼57–58 kDa), whereas the 33- and 38-kDa products may represent the smaller PTBP2 isoforms 5 and/or 6 (predicted size ∼38 kDa). The 36-kDa product detected by the anti-SRSF10 antibody may represent SRSF10 isoforms 1 and/or 2 (predicted size ∼31 kDa), whereas the 20- to 25-kDa products may represent isoforms 3, 4, and/or 5 (predicted size ∼20–22 kDa). Overexpression of SRSF10 alone or in combination with PTBP2 resulted in significantly reduced expression of canonical human DEAF1 ( D ), increased expression of DEAF1-VAR1 ( E ), and reduced expression of the PTA gene CELA1 ( F ). Overexpression of PTBP2 resulted only in the increased expression of DEAF1-VAR1 48 h after transfection ( E ). G : A schematic diagram showing how inflammation and hyperglycemia may contribute to reduced DEAF1 function and reduced PTA expression in LNSCs during the progression of disease. Data shown in A , B , and D–F represent the means ± SEM of at least 3 independent experiments performed in triplicate. C shows data that are representative of 4 separate experiments. All QPCR data were normalized to 18S rRNA expression. Statistical analysis was performed using the two-tailed unpaired Student t test. * P
    Figure Legend Snippet: PTBP2 and SRSF10 mediate the splicing of human DEAF1 . A–F : QPCR and immunoblotting data showing transfection of HEK293T cells with plasmids expressing PTBP2 or SRSF10 , alone (1 μg each) or in combination (0.5 μg each). Transfection resulted in significantly increased mRNA ( A and B ) and protein ( C ) expression of the corresponding genes 24 and 48 h after transfection. C : The antibodies used detected multiple alternatively spliced isoforms of PTBP2 and SRSF10. The ∼57-kDa product detected by the anti-PTBP2 antibody may represent the larger PTBP2 isoforms 1, 2, 3, and/or 4 (predicted size ∼57–58 kDa), whereas the 33- and 38-kDa products may represent the smaller PTBP2 isoforms 5 and/or 6 (predicted size ∼38 kDa). The 36-kDa product detected by the anti-SRSF10 antibody may represent SRSF10 isoforms 1 and/or 2 (predicted size ∼31 kDa), whereas the 20- to 25-kDa products may represent isoforms 3, 4, and/or 5 (predicted size ∼20–22 kDa). Overexpression of SRSF10 alone or in combination with PTBP2 resulted in significantly reduced expression of canonical human DEAF1 ( D ), increased expression of DEAF1-VAR1 ( E ), and reduced expression of the PTA gene CELA1 ( F ). Overexpression of PTBP2 resulted only in the increased expression of DEAF1-VAR1 48 h after transfection ( E ). G : A schematic diagram showing how inflammation and hyperglycemia may contribute to reduced DEAF1 function and reduced PTA expression in LNSCs during the progression of disease. Data shown in A , B , and D–F represent the means ± SEM of at least 3 independent experiments performed in triplicate. C shows data that are representative of 4 separate experiments. All QPCR data were normalized to 18S rRNA expression. Statistical analysis was performed using the two-tailed unpaired Student t test. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection, Expressing, Over Expression, Two Tailed Test

    7) Product Images from "Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus"

    Article Title: Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus

    Journal: Molecular Brain

    doi: 10.1186/s13041-015-0159-1

    cAMP-activated AVP promoter activity is modulated through Creb3l1. a - b AtT20 cells were transfected with 1 kb rat Avp promoter construct. At 24 h after transfection the cells were a treated with 10 μM FSK for 4 and 24 h or b pre-treated with 100nM DEX or DMSO for 2 h and followed by 4 h treatment of 10 μM FSK or DMSO and luciferase activity was measured ( n = 3). c – e The Creb3l1 -knockdown AtT20 cell line was produced in parallel with a control non-targeting shRNA cell line by transduction of lentivirus containing Creb3l1 or control non-targeting shRNA and further selected with puromycin. c The level of Creb3l1 knockdown was investigated by qPCR and immunoblotting in the AtT20 shRNA cell lines ( n = 3). d The effect of FSK on Creb3l1 mRNA expression was examined by qPCR in control non-targeting and Creb3l1 knockdown cell lines ( n = 3). e Luciferase assays were performed in control and Creb3l1 -knockdown cell lines using 1 kb Avp promoter construct driving luciferase reporter gene ( n = 3-4). Error bar + SEM; *, p
    Figure Legend Snippet: cAMP-activated AVP promoter activity is modulated through Creb3l1. a - b AtT20 cells were transfected with 1 kb rat Avp promoter construct. At 24 h after transfection the cells were a treated with 10 μM FSK for 4 and 24 h or b pre-treated with 100nM DEX or DMSO for 2 h and followed by 4 h treatment of 10 μM FSK or DMSO and luciferase activity was measured ( n = 3). c – e The Creb3l1 -knockdown AtT20 cell line was produced in parallel with a control non-targeting shRNA cell line by transduction of lentivirus containing Creb3l1 or control non-targeting shRNA and further selected with puromycin. c The level of Creb3l1 knockdown was investigated by qPCR and immunoblotting in the AtT20 shRNA cell lines ( n = 3). d The effect of FSK on Creb3l1 mRNA expression was examined by qPCR in control non-targeting and Creb3l1 knockdown cell lines ( n = 3). e Luciferase assays were performed in control and Creb3l1 -knockdown cell lines using 1 kb Avp promoter construct driving luciferase reporter gene ( n = 3-4). Error bar + SEM; *, p

    Techniques Used: Activity Assay, Transfection, Construct, Luciferase, Produced, shRNA, Transduction, Real-time Polymerase Chain Reaction, Expressing

    cAMP and glucocorticoid regulate Creb3l1 expression. AtT20 cells were used to study regulation of Creb3l1 . a – c AtT20 cells were treated with 10 μM FSK or vehicle (DMSO). a Creb3l1 mRNA expression was observed by qPCR at various time points ( n = 3; One way ANOVA). b – c Immunoblotting was performed to examine protein expression level of CREB3L1 in both b total protein extracts and c cytosolic/nuclear extracts. d – e AtT20 cells were treated with vehicle (DMSO), 10 μM FSK and/or 100nM DEX at various time points. d Effect of glucocorticoid on c-Fos and Creb3l1 expression was examined by qPCR ( n = 3; Two way ANOVA). e Immunoblotting of CREB3L1 was performed at 24 h after treatment. GAPDH was used as an internal control for immunoblotting. * in B and E indicates non-specific band in immunoblotting. * in D indicate significant from FSK + DEX compared to FSK group. Scale bar, 20 μm; Error bar, +SEM; *, p
    Figure Legend Snippet: cAMP and glucocorticoid regulate Creb3l1 expression. AtT20 cells were used to study regulation of Creb3l1 . a – c AtT20 cells were treated with 10 μM FSK or vehicle (DMSO). a Creb3l1 mRNA expression was observed by qPCR at various time points ( n = 3; One way ANOVA). b – c Immunoblotting was performed to examine protein expression level of CREB3L1 in both b total protein extracts and c cytosolic/nuclear extracts. d – e AtT20 cells were treated with vehicle (DMSO), 10 μM FSK and/or 100nM DEX at various time points. d Effect of glucocorticoid on c-Fos and Creb3l1 expression was examined by qPCR ( n = 3; Two way ANOVA). e Immunoblotting of CREB3L1 was performed at 24 h after treatment. GAPDH was used as an internal control for immunoblotting. * in B and E indicates non-specific band in immunoblotting. * in D indicate significant from FSK + DEX compared to FSK group. Scale bar, 20 μm; Error bar, +SEM; *, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    8) Product Images from "MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases"

    Article Title: MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms161025377

    AS and HSA cell lines over-secreted miR-214 and miR-126 via MVs. ( A ) Immunoblotting for MV-markers: CD63 and CD81. The nanoparticles from all cell lines expressed CD63 or CD81 although CD81 was absent or weakly detected in MVs derived from HAMON, CnAOEC, JuB2, and Re12. β-actin was used as a negative control for excluding contamination by cellular contents; ( B ) Nanoparticle tracking analysis for AS and HSA cell lines and control ECs. The peaks of MV diameters showed a range of 144–269 nm, indicating that AS, HSA cell lines and control ECs mostly secreted MVs having a diameter of over 100 nm; ( C ) Quantitative measurement of miR-214 and miR-126 in the MVs from the conditioned media from AS, HSA cell lines and control ECs. The levels of miR-214 were significantly increased in the conditioned media of AS and HSA cell lines compared with those for the control cell lines. The levels of miR-126 were also increased likewise, except in the case of ISO-HAS. The degree of increase in canine cell lines was dramatically higher than that in human cell lines. All data are presented as the mean of triplicate experiments with error bars indicating the s.e.m. (Unpaired two-tailed t -test; * p
    Figure Legend Snippet: AS and HSA cell lines over-secreted miR-214 and miR-126 via MVs. ( A ) Immunoblotting for MV-markers: CD63 and CD81. The nanoparticles from all cell lines expressed CD63 or CD81 although CD81 was absent or weakly detected in MVs derived from HAMON, CnAOEC, JuB2, and Re12. β-actin was used as a negative control for excluding contamination by cellular contents; ( B ) Nanoparticle tracking analysis for AS and HSA cell lines and control ECs. The peaks of MV diameters showed a range of 144–269 nm, indicating that AS, HSA cell lines and control ECs mostly secreted MVs having a diameter of over 100 nm; ( C ) Quantitative measurement of miR-214 and miR-126 in the MVs from the conditioned media from AS, HSA cell lines and control ECs. The levels of miR-214 were significantly increased in the conditioned media of AS and HSA cell lines compared with those for the control cell lines. The levels of miR-126 were also increased likewise, except in the case of ISO-HAS. The degree of increase in canine cell lines was dramatically higher than that in human cell lines. All data are presented as the mean of triplicate experiments with error bars indicating the s.e.m. (Unpaired two-tailed t -test; * p

    Techniques Used: Derivative Assay, Negative Control, Two Tailed Test

    9) Product Images from "A phospholipid transfer function of ER-mitochondria encounter structure revealed in vitro"

    Article Title: A phospholipid transfer function of ER-mitochondria encounter structure revealed in vitro

    Journal: Scientific Reports

    doi: 10.1038/srep30777

    Fermentable and low-nutrient conditions are suitable for isolation of membrane fractions containing both the ER and mitochondria. ( A ) Phospholipid biosynthetic pathways in the ER and mitochondria. PA, phosphatidic acid; CDP-DAG, CDP-diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; OM, mitochondrial outer membrane; IMS, intermembrane space; IM, mitochondrial inner membrane. ( B ) The heavy membrane fractions were incubated with [ 14 C]-serine in the presence or absence of 2 mM CTP at 30 °C for the indicated time. Total phospholipids were extracted and analyzed by TLC and radioimaging. The total amount of synthesized phospholipid (PS+PE) in the absence of CTP after 30 min incubation was set to 100%. ( C ) PS was synthesized in vitro using the same membrane fractions as in ( B ) in the presence of CTP and S-adenosylmethionine. ( D ) Yeast cells cultivated in YPLac, YPD or SCD were subjected to subcellular fractionation. 12 k, 25 k and 40 k pellets were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies against phospholipid synthetic enzymes, ER and mitochondrial marker proteins. ( E ) The 12 k pellet fractions isolated from yeast cells cultivated in YPLac, YPD or SCD were incubated with [ 14 C]-serine for the indicated times. Total phospholipids were extracted and analyzed by TLC and radioimaging. ( F ) 12 k pellet prepared from wild-type cells or mutant cells lacking a phospholipid synthetic enzyme were incubated with [ 14 C]-serine and synthesized radiolabeled phospholipids were analyzed as in ( B ).
    Figure Legend Snippet: Fermentable and low-nutrient conditions are suitable for isolation of membrane fractions containing both the ER and mitochondria. ( A ) Phospholipid biosynthetic pathways in the ER and mitochondria. PA, phosphatidic acid; CDP-DAG, CDP-diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; OM, mitochondrial outer membrane; IMS, intermembrane space; IM, mitochondrial inner membrane. ( B ) The heavy membrane fractions were incubated with [ 14 C]-serine in the presence or absence of 2 mM CTP at 30 °C for the indicated time. Total phospholipids were extracted and analyzed by TLC and radioimaging. The total amount of synthesized phospholipid (PS+PE) in the absence of CTP after 30 min incubation was set to 100%. ( C ) PS was synthesized in vitro using the same membrane fractions as in ( B ) in the presence of CTP and S-adenosylmethionine. ( D ) Yeast cells cultivated in YPLac, YPD or SCD were subjected to subcellular fractionation. 12 k, 25 k and 40 k pellets were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies against phospholipid synthetic enzymes, ER and mitochondrial marker proteins. ( E ) The 12 k pellet fractions isolated from yeast cells cultivated in YPLac, YPD or SCD were incubated with [ 14 C]-serine for the indicated times. Total phospholipids were extracted and analyzed by TLC and radioimaging. ( F ) 12 k pellet prepared from wild-type cells or mutant cells lacking a phospholipid synthetic enzyme were incubated with [ 14 C]-serine and synthesized radiolabeled phospholipids were analyzed as in ( B ).

    Techniques Used: Isolation, Incubation, Thin Layer Chromatography, Synthesized, In Vitro, Fractionation, SDS Page, Marker, Mutagenesis

    ERMES facilitates phospholipid transport between the ER and mitochondria. ( A ) Proteins in the heavy membrane fractions isolated from wild-type, mmm1∆ and mdm12∆ cells expressing Vps13-D716H were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies. The asterisk indicates a nonspecific band. ( B ) In vitro PS transport assays were performed using the heavy membrane fractions isolated from wild-type, mmm1∆ and mdm12∆ cells expressing Vps13-D716H. ( C ) Amounts of PS, PE and PDME+PC relative to total phospholipids were calculated and plotted. Values are mean ± SEM ( n = 4). The amount of total phospholipids synthesized with wild-type cells after 40 min incubation was set to 100% (Total).
    Figure Legend Snippet: ERMES facilitates phospholipid transport between the ER and mitochondria. ( A ) Proteins in the heavy membrane fractions isolated from wild-type, mmm1∆ and mdm12∆ cells expressing Vps13-D716H were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies. The asterisk indicates a nonspecific band. ( B ) In vitro PS transport assays were performed using the heavy membrane fractions isolated from wild-type, mmm1∆ and mdm12∆ cells expressing Vps13-D716H. ( C ) Amounts of PS, PE and PDME+PC relative to total phospholipids were calculated and plotted. Values are mean ± SEM ( n = 4). The amount of total phospholipids synthesized with wild-type cells after 40 min incubation was set to 100% (Total).

    Techniques Used: Isolation, Expressing, SDS Page, In Vitro, Synthesized, Incubation

    10) Product Images from "A Conserved Motif in the ITK PH-Domain Is Required for Phosphoinositide Binding and TCR Signaling but Dispensable for Adaptor Protein Interactions"

    Article Title: A Conserved Motif in the ITK PH-Domain Is Required for Phosphoinositide Binding and TCR Signaling but Dispensable for Adaptor Protein Interactions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045158

    FYF-mutant ITK fails to bind PIP 3 . Whole cell lysates (WCL) obtained from HEK-293T cells transfected with untagged WT- or FYF-ITK mutant were resolved by SDS-PAGE and analyzed by immunoblotting with anti-ITK antibodies (left panels). An aliquot of each lysate was incubated with PIP 3 -coated beads followed by washing, elution, and SDS-PAGE/immunoblot analysis for ITK content (right panels). Results are representative of three independent experiments.
    Figure Legend Snippet: FYF-mutant ITK fails to bind PIP 3 . Whole cell lysates (WCL) obtained from HEK-293T cells transfected with untagged WT- or FYF-ITK mutant were resolved by SDS-PAGE and analyzed by immunoblotting with anti-ITK antibodies (left panels). An aliquot of each lysate was incubated with PIP 3 -coated beads followed by washing, elution, and SDS-PAGE/immunoblot analysis for ITK content (right panels). Results are representative of three independent experiments.

    Techniques Used: Mutagenesis, Transfection, SDS Page, Incubation

    11) Product Images from "A Conserved Motif in the ITK PH-Domain Is Required for Phosphoinositide Binding and TCR Signaling but Dispensable for Adaptor Protein Interactions"

    Article Title: A Conserved Motif in the ITK PH-Domain Is Required for Phosphoinositide Binding and TCR Signaling but Dispensable for Adaptor Protein Interactions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045158

    FYF-mutant ITK fails to bind PIP 3 . Whole cell lysates (WCL) obtained from HEK-293T cells transfected with untagged WT- or FYF-ITK mutant were resolved by SDS-PAGE and analyzed by immunoblotting with anti-ITK antibodies (left panels). An aliquot of each lysate was incubated with PIP 3 -coated beads followed by washing, elution, and SDS-PAGE/immunoblot analysis for ITK content (right panels). Results are representative of three independent experiments.
    Figure Legend Snippet: FYF-mutant ITK fails to bind PIP 3 . Whole cell lysates (WCL) obtained from HEK-293T cells transfected with untagged WT- or FYF-ITK mutant were resolved by SDS-PAGE and analyzed by immunoblotting with anti-ITK antibodies (left panels). An aliquot of each lysate was incubated with PIP 3 -coated beads followed by washing, elution, and SDS-PAGE/immunoblot analysis for ITK content (right panels). Results are representative of three independent experiments.

    Techniques Used: Mutagenesis, Transfection, SDS Page, Incubation

    12) Product Images from "TRIM25 blockade by RNA interference inhibited migration and invasion of gastric cancer cells through TGF-β signaling"

    Article Title: TRIM25 blockade by RNA interference inhibited migration and invasion of gastric cancer cells through TGF-β signaling

    Journal: Scientific Reports

    doi: 10.1038/srep19070

    TRIM25 expression was suppressed by siRNA transfection in GC cells. Expression of TRIM25 in MGC-803 ( A ) and AGS cells ( B ) was analyzed by qRT-PCR (left panel) and immunoblotting (middle and right panels). WT: wild type cells; siNC: scrambled siRNA-transfected cells; siTRIM25-1, siTRIM25-2: TRIM25-siRNA-1 or TRIM25-siRNA-2 transfected cells (* P
    Figure Legend Snippet: TRIM25 expression was suppressed by siRNA transfection in GC cells. Expression of TRIM25 in MGC-803 ( A ) and AGS cells ( B ) was analyzed by qRT-PCR (left panel) and immunoblotting (middle and right panels). WT: wild type cells; siNC: scrambled siRNA-transfected cells; siTRIM25-1, siTRIM25-2: TRIM25-siRNA-1 or TRIM25-siRNA-2 transfected cells (* P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    Ectopic expression of TRIM25 reversed the effects of TGF-β inhibitor on the migration and invasion of MKN-28 cells. ( A ) MKN-28 cells was transfected with control plasmid (NC) or expression plasmid encoding TRIM25. 48 h after transfection, expression of TRIM25 was analyzed by qRT-PCR and immunoblotting. ( B,C ) MKN-28 cells transfected with TRIM25 or NC, and migration and invasion assay were performed in a transwell chamber containing either DMSO or 10 μM TGF-β inhibitor, SB431542 (Sigma). Scale bar: 100 μm.
    Figure Legend Snippet: Ectopic expression of TRIM25 reversed the effects of TGF-β inhibitor on the migration and invasion of MKN-28 cells. ( A ) MKN-28 cells was transfected with control plasmid (NC) or expression plasmid encoding TRIM25. 48 h after transfection, expression of TRIM25 was analyzed by qRT-PCR and immunoblotting. ( B,C ) MKN-28 cells transfected with TRIM25 or NC, and migration and invasion assay were performed in a transwell chamber containing either DMSO or 10 μM TGF-β inhibitor, SB431542 (Sigma). Scale bar: 100 μm.

    Techniques Used: Expressing, Migration, Transfection, Plasmid Preparation, Quantitative RT-PCR, Invasion Assay

    TRIM25 expression affected migration, E-cadherin and TGF-β signaling pathways. ( A ) GSEA was performed using TCGA dataset. TRIM25-higher expression was closely related with migration, E-cadherin and TGF-β signaling pathways. ( B,C ) Expression of the above three pathway related gene was evaluated by immunoblotting in MGC-803 and AGS cells at 48 h after transfected with TRIM25 siRNA-1 or control siRNA. ( D,E ) Phosphorylation of Smad2 and Smad4 was evaluated at 6 h after transfection by immunoblotting. WT: wild type cells; siNC: scrambled siRNA-transfected cells; siTRIM25-1: TRIM25-siRNA-1-transfected cells (* P
    Figure Legend Snippet: TRIM25 expression affected migration, E-cadherin and TGF-β signaling pathways. ( A ) GSEA was performed using TCGA dataset. TRIM25-higher expression was closely related with migration, E-cadherin and TGF-β signaling pathways. ( B,C ) Expression of the above three pathway related gene was evaluated by immunoblotting in MGC-803 and AGS cells at 48 h after transfected with TRIM25 siRNA-1 or control siRNA. ( D,E ) Phosphorylation of Smad2 and Smad4 was evaluated at 6 h after transfection by immunoblotting. WT: wild type cells; siNC: scrambled siRNA-transfected cells; siTRIM25-1: TRIM25-siRNA-1-transfected cells (* P

    Techniques Used: Expressing, Migration, Transfection

    Ectopic expression of TRIM25 reversed the effects of TGF-β inhibitor on TGF-β signaling. ( A ) Expression of MMP-2 and MMP-9 was evaluated by immunoblotting. ( B ) Phosphorylation of Smad2 and Smad4 was determined by immunoblotting (* P
    Figure Legend Snippet: Ectopic expression of TRIM25 reversed the effects of TGF-β inhibitor on TGF-β signaling. ( A ) Expression of MMP-2 and MMP-9 was evaluated by immunoblotting. ( B ) Phosphorylation of Smad2 and Smad4 was determined by immunoblotting (* P

    Techniques Used: Expressing

    13) Product Images from "The Png1-Rad23 complex regulates glycoprotein turnover"

    Article Title: The Png1-Rad23 complex regulates glycoprotein turnover

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200507149

    Degradation of RTA in various yeast mutants. (A) Expressed RTA proteins exist in both g1 and g0 forms in yeast. Flag-tagged RTA was overexpressed in wild-type or sec65-1 mutant yeast cells and recovered by immunoprecipitation. RTA immunoprecipitates were mock treated (−) or digested (+) with EndoH, resolved by SDS-PAGE, and visualized by immunoblotting. The arrows indicate RTA proteins modified with one (g1) or no (g0) glycan. In sec65-1 cells (lanes 3 and 4), untranslocated RTA containing its ER signal sequence was also detected and is indicated by an asterisk. (B) Involvement of known ERAD components in RTA degradation. Wild-type (BY4741) and mutant cells containing a GAL1 promoter–regulated Flag-RTA were first grown in raffinose-containing media. Expression of RTA was induced by the addition of galactose. Samples were taken after promoter shutoff at intervals and analyzed by anti-Flag Western blots. Equal amounts of protein extracts were used and confirmed by blotting with anti-Rpt5 antibody in all the promoter shutoff experiments (not depicted). (C) Efficient degradation of glycosylated RTA requires Rad23 and Png1. Pulse-chase analysis of RTA in wild-type and mutant cells was performed as described in Material and methods. (D–F) Quantitation of the data in C for glycosylated (g1) RTA, g0 RTA, and the combined intensity of both g1 and g0 RTA. The amount of proteins was determined by phosphorimager analysis.
    Figure Legend Snippet: Degradation of RTA in various yeast mutants. (A) Expressed RTA proteins exist in both g1 and g0 forms in yeast. Flag-tagged RTA was overexpressed in wild-type or sec65-1 mutant yeast cells and recovered by immunoprecipitation. RTA immunoprecipitates were mock treated (−) or digested (+) with EndoH, resolved by SDS-PAGE, and visualized by immunoblotting. The arrows indicate RTA proteins modified with one (g1) or no (g0) glycan. In sec65-1 cells (lanes 3 and 4), untranslocated RTA containing its ER signal sequence was also detected and is indicated by an asterisk. (B) Involvement of known ERAD components in RTA degradation. Wild-type (BY4741) and mutant cells containing a GAL1 promoter–regulated Flag-RTA were first grown in raffinose-containing media. Expression of RTA was induced by the addition of galactose. Samples were taken after promoter shutoff at intervals and analyzed by anti-Flag Western blots. Equal amounts of protein extracts were used and confirmed by blotting with anti-Rpt5 antibody in all the promoter shutoff experiments (not depicted). (C) Efficient degradation of glycosylated RTA requires Rad23 and Png1. Pulse-chase analysis of RTA in wild-type and mutant cells was performed as described in Material and methods. (D–F) Quantitation of the data in C for glycosylated (g1) RTA, g0 RTA, and the combined intensity of both g1 and g0 RTA. The amount of proteins was determined by phosphorimager analysis.

    Techniques Used: Mutagenesis, Immunoprecipitation, SDS Page, Modification, Sequencing, Expressing, Western Blot, Pulse Chase, Quantitation Assay

    14) Product Images from "Systemic Exposure to Air Pollution Induces Oxidative Stress and Inflammation in Mouse Brain, Contributing to Neurodegeneration Onset"

    Article Title: Systemic Exposure to Air Pollution Induces Oxidative Stress and Inflammation in Mouse Brain, Contributing to Neurodegeneration Onset

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21103699

    Amyloidogenic precursor protein (APP) processing analysis after single and repeated instillations of BB and DEP. Representative immunoblotting images of amyloid precursor protein (APP), phosphorylated APP on threonine 668 (p-APP Thr668 ), and beta-secretase 1 (BACE1) analysis in mice after single ( A ) and repeated ( E ) instillations with 50 µg of BB or DEP/100 µL 0.9% NaCl. Histograms display p-APP Thr668 /APP, APP, and BACE1 protein levels in mice after single ( B – D ) and repeated ( F – H ) instillations with BB and DEP, with respect to sham. Proteins are normalized to corresponding total proteins revealed by Ponceau in each lane ( Figure S3, Supplementary Materials ), and the data are expressed as means ± SEM ( n = 6). Statistical differences were tested accordingly by one-way ANOVA followed by Tukey post hoc comparison. ** p
    Figure Legend Snippet: Amyloidogenic precursor protein (APP) processing analysis after single and repeated instillations of BB and DEP. Representative immunoblotting images of amyloid precursor protein (APP), phosphorylated APP on threonine 668 (p-APP Thr668 ), and beta-secretase 1 (BACE1) analysis in mice after single ( A ) and repeated ( E ) instillations with 50 µg of BB or DEP/100 µL 0.9% NaCl. Histograms display p-APP Thr668 /APP, APP, and BACE1 protein levels in mice after single ( B – D ) and repeated ( F – H ) instillations with BB and DEP, with respect to sham. Proteins are normalized to corresponding total proteins revealed by Ponceau in each lane ( Figure S3, Supplementary Materials ), and the data are expressed as means ± SEM ( n = 6). Statistical differences were tested accordingly by one-way ANOVA followed by Tukey post hoc comparison. ** p

    Techniques Used: Mouse Assay

    Inflammation analysis after single and repeated instillations of BB and DEP. ( A – F ) Representative immunoblotting images of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 ( COX-2) analysis in mice after single ( A ) and repeated ( D ) instillations with 50 µg of BB or DEP/100 µL 0.9% NaCl. Histograms display iNOS and COX-2 expression in mice after single ( B , C ) and repeated ( E , F ) instillations with BB and DEP, with respect to sham. Proteins are normalized to corresponding total proteins revealed by Ponceau in each lane ( Figure S3, Supplementary Materials ), and the data are expressed as means ± SEM ( n = 6). Statistical differences were tested accordingly by one-way ANOVA followed by Tukey post hoc comparison. * p
    Figure Legend Snippet: Inflammation analysis after single and repeated instillations of BB and DEP. ( A – F ) Representative immunoblotting images of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 ( COX-2) analysis in mice after single ( A ) and repeated ( D ) instillations with 50 µg of BB or DEP/100 µL 0.9% NaCl. Histograms display iNOS and COX-2 expression in mice after single ( B , C ) and repeated ( E , F ) instillations with BB and DEP, with respect to sham. Proteins are normalized to corresponding total proteins revealed by Ponceau in each lane ( Figure S3, Supplementary Materials ), and the data are expressed as means ± SEM ( n = 6). Statistical differences were tested accordingly by one-way ANOVA followed by Tukey post hoc comparison. * p

    Techniques Used: Mouse Assay, Expressing

    Oxidative stress analysis after single and repeated instillations of biomass burning-derived (BB) particles and diesel exhaust particles (DEP). Representative immunoblotting images of heme oxygenase-1 (HO-1), heat-shock protein 70 (Hsp70), and Cytochrome P450 1b1 (Cyp1b1) analysis in mice after single ( A ) and repeated ( E ) instillations with 50 µg of BB or DEP/100 µL 0.9% NaCl. Histograms display HO-1, Hsp70, and Cyp1b1 expression in mice after single ( B – D ) and repeated ( F – H ) instillations with BB and DEP, with respect to sham. Proteins are normalized to corresponding total proteins revealed by Ponceau in each lane ( Figure S3, Supplementary Materials ), and the data are expressed as means ± standard error of the mean (SEM) ( n = 6). Statistical differences were tested accordingly by one-way ANOVA followed by Tukey post hoc comparison. * p
    Figure Legend Snippet: Oxidative stress analysis after single and repeated instillations of biomass burning-derived (BB) particles and diesel exhaust particles (DEP). Representative immunoblotting images of heme oxygenase-1 (HO-1), heat-shock protein 70 (Hsp70), and Cytochrome P450 1b1 (Cyp1b1) analysis in mice after single ( A ) and repeated ( E ) instillations with 50 µg of BB or DEP/100 µL 0.9% NaCl. Histograms display HO-1, Hsp70, and Cyp1b1 expression in mice after single ( B – D ) and repeated ( F – H ) instillations with BB and DEP, with respect to sham. Proteins are normalized to corresponding total proteins revealed by Ponceau in each lane ( Figure S3, Supplementary Materials ), and the data are expressed as means ± standard error of the mean (SEM) ( n = 6). Statistical differences were tested accordingly by one-way ANOVA followed by Tukey post hoc comparison. * p

    Techniques Used: Derivative Assay, Mouse Assay, Expressing

    15) Product Images from "Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state"

    Article Title: Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state

    Journal: bioRxiv

    doi: 10.1101/2020.05.14.096891

    glycine-95 regulates oligomerization of IFITM3 in bulk cell lysates. (A) HEK293T were transiently transfected with empty pQCXIP or the following pairs: IFITM3-FLAG and IFITM3-myc or G95L-FLAG and G95L-myc. Whole cell lysates were produced under mildly denaturing conditions and immunoprecipitation (IP) using anti-FLAG antibody was performed. IP fractions and volumes of whole cell lysates were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti-FLAG and anti-myc. Number and tick marks indicate size (kilodaltons) and position of protein standards in ladder. (B) Levels of IFITM3-myc (either WT or G95L) co-immunoprecipitated by anti-FLAG IP were quantified from conditions in (A). Results represent the mean of three independent experiments. Error bars indicate standard error. (C) HEK293T were transiently transfected with empty pQCXIP, WT IFITM3-FLAG or G95L-FLAG. Cell lysates were produced with 1% digitonin and blue native PAGE was performed, followed by immunoblotting with anti-FLAG. Number and tick marks indicate size (kilodaltons) and position of protein standards in ladder. Statistical analysis was performed using student’s T test. *, P
    Figure Legend Snippet: glycine-95 regulates oligomerization of IFITM3 in bulk cell lysates. (A) HEK293T were transiently transfected with empty pQCXIP or the following pairs: IFITM3-FLAG and IFITM3-myc or G95L-FLAG and G95L-myc. Whole cell lysates were produced under mildly denaturing conditions and immunoprecipitation (IP) using anti-FLAG antibody was performed. IP fractions and volumes of whole cell lysates were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti-FLAG and anti-myc. Number and tick marks indicate size (kilodaltons) and position of protein standards in ladder. (B) Levels of IFITM3-myc (either WT or G95L) co-immunoprecipitated by anti-FLAG IP were quantified from conditions in (A). Results represent the mean of three independent experiments. Error bars indicate standard error. (C) HEK293T were transiently transfected with empty pQCXIP, WT IFITM3-FLAG or G95L-FLAG. Cell lysates were produced with 1% digitonin and blue native PAGE was performed, followed by immunoblotting with anti-FLAG. Number and tick marks indicate size (kilodaltons) and position of protein standards in ladder. Statistical analysis was performed using student’s T test. *, P

    Techniques Used: Transfection, Produced, Immunoprecipitation, SDS Page, Western Blot, Blue Native PAGE

    Expression, subcellular localization, and anti-HIV activities of WT and mutant IFITM3. (A) SDS-PAGE and Western blot analysis of whole cell lysates produced from HEK293T transiently transfected with empty pQCXIP, WT IFITM3-FLAG, or mutants. Immunoblotting was performed with anti-FLAG. Actin served as a loading control. Number and tick marks indicate size (kilodaltons) and position of protein standards in ladder. (B) HEK293T cells were co-transfected with EEA1-GFP and empty pQCXIP or IFITM3-FLAG (encoding WT or G95L). Cells were fixed, permeabilized, and immunostained at 48 hours post-transfection with anti-IFITM3 and anti-CD63 and cells were analyzed by immunofluorescence confocal microscopy. All images are average Z-stacks from 10 consecutive medial sections. Scale bars, 10 μm. (C) Whole cell lysates and virus-containing supernatants were collected from HEK293T co-transfected with the HIV-1 molecular clone NL4.3 and empty pQCXIP, WT IFITM3-FLAG, or the indicated mutant at 48 hours post-transfection. Virus-containing supernatants were ultracentrifuged through sucrose cushions. Both lysates and concentrated, purified virus-containing supernatants were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti-gp120, anti-gp41, anti-CA, anti-actin, and anti-FLAG. Levels of IFITM3 (FLAG), gp120, and gp41 were quantified and normalized to levels of CA. For anti-FLAG immunoblotting, the amount of WT IFITM3 in virions was set to 100%. For anti-gp120 and anti-gp41, levels observed in the pQCXIP empty vector were set to 100%.
    Figure Legend Snippet: Expression, subcellular localization, and anti-HIV activities of WT and mutant IFITM3. (A) SDS-PAGE and Western blot analysis of whole cell lysates produced from HEK293T transiently transfected with empty pQCXIP, WT IFITM3-FLAG, or mutants. Immunoblotting was performed with anti-FLAG. Actin served as a loading control. Number and tick marks indicate size (kilodaltons) and position of protein standards in ladder. (B) HEK293T cells were co-transfected with EEA1-GFP and empty pQCXIP or IFITM3-FLAG (encoding WT or G95L). Cells were fixed, permeabilized, and immunostained at 48 hours post-transfection with anti-IFITM3 and anti-CD63 and cells were analyzed by immunofluorescence confocal microscopy. All images are average Z-stacks from 10 consecutive medial sections. Scale bars, 10 μm. (C) Whole cell lysates and virus-containing supernatants were collected from HEK293T co-transfected with the HIV-1 molecular clone NL4.3 and empty pQCXIP, WT IFITM3-FLAG, or the indicated mutant at 48 hours post-transfection. Virus-containing supernatants were ultracentrifuged through sucrose cushions. Both lysates and concentrated, purified virus-containing supernatants were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti-gp120, anti-gp41, anti-CA, anti-actin, and anti-FLAG. Levels of IFITM3 (FLAG), gp120, and gp41 were quantified and normalized to levels of CA. For anti-FLAG immunoblotting, the amount of WT IFITM3 in virions was set to 100%. For anti-gp120 and anti-gp41, levels observed in the pQCXIP empty vector were set to 100%.

    Techniques Used: Expressing, Mutagenesis, SDS Page, Western Blot, Produced, Transfection, Immunofluorescence, Confocal Microscopy, Purification, Plasmid Preparation

    16) Product Images from "Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs"

    Article Title: Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.972950

    Phosphorylation of RtcB on Y306 negatively affects XBP1 mRNA splicing but not RIDD activity. ( A, D ) HeLa lines stably expressing wt or mutant RtcB-Flag were treated with 1μg/ml Tun for 0, 2, 4, 8, 16 and 24 hours. cDNA coming from these were analyzed by qPCR for XBP1s and XBP1 total mRNA levels with their ratio being the XBP1 mRNA splicing as depicted in the y axis of the graphs. The Parental HeLa cells are also included for comparison. ( B, E ) The peak of XBP1s activity at 8 hours (A, D) was then normalized to the protein levels (C, F) to obtain the RtcB specific activity. ( C, F ) Stable HeLa lines were lysed and analyzed for RtcB-Flag expression levels by immunoblotting with anti-Flag. Actin was used as a loading control. ( G ) HeLa lines stably expressing wt or Y306F RtcB-Flag were left untransfected or transfected with an siRNA targeting the 3’-UTR of the RTCB mRNA. 48 hours post-transfection the cells were pre-treated for 2 hours with 5μg/ml Actinomycin D and then treated either with 5μg/ml Tun for 4 hours or 1mM DTT for 2 hours. The graph shows the mRNA levels of PER1 (upper part) and SCARA3 (lower part) after normalization to the untreated samples (0h time-point). ( H ) RT-qPCR analysis of the untreated samples from (G) for the endogenous RTCB mRNA using primers spanning its 3’-UTR. ( I ) cDNA from HeLa stable lines expressing wt or Y306F mutant RtcB-Flag was analyzed with qPCR for the splicing of the three intron-containing tRNA molecules in human Tyr, Arg and Ile. The graph shows the percentage of each spliced tRNA molecule to their total levels (unspliced + spliced). The presented values have been normalized to the levels of % tRNA splicing in Parental HeLa cells. ( J ) The levels of the intronless tRNA molecules Pro and Val were measured by RT-qPCR in the samples of (I). Values have been normalized to the levels measured in Parental HeLa cells. Data values for all the presented graphs are the mean ± SEM of n≥3 independent experiments (*p
    Figure Legend Snippet: Phosphorylation of RtcB on Y306 negatively affects XBP1 mRNA splicing but not RIDD activity. ( A, D ) HeLa lines stably expressing wt or mutant RtcB-Flag were treated with 1μg/ml Tun for 0, 2, 4, 8, 16 and 24 hours. cDNA coming from these were analyzed by qPCR for XBP1s and XBP1 total mRNA levels with their ratio being the XBP1 mRNA splicing as depicted in the y axis of the graphs. The Parental HeLa cells are also included for comparison. ( B, E ) The peak of XBP1s activity at 8 hours (A, D) was then normalized to the protein levels (C, F) to obtain the RtcB specific activity. ( C, F ) Stable HeLa lines were lysed and analyzed for RtcB-Flag expression levels by immunoblotting with anti-Flag. Actin was used as a loading control. ( G ) HeLa lines stably expressing wt or Y306F RtcB-Flag were left untransfected or transfected with an siRNA targeting the 3’-UTR of the RTCB mRNA. 48 hours post-transfection the cells were pre-treated for 2 hours with 5μg/ml Actinomycin D and then treated either with 5μg/ml Tun for 4 hours or 1mM DTT for 2 hours. The graph shows the mRNA levels of PER1 (upper part) and SCARA3 (lower part) after normalization to the untreated samples (0h time-point). ( H ) RT-qPCR analysis of the untreated samples from (G) for the endogenous RTCB mRNA using primers spanning its 3’-UTR. ( I ) cDNA from HeLa stable lines expressing wt or Y306F mutant RtcB-Flag was analyzed with qPCR for the splicing of the three intron-containing tRNA molecules in human Tyr, Arg and Ile. The graph shows the percentage of each spliced tRNA molecule to their total levels (unspliced + spliced). The presented values have been normalized to the levels of % tRNA splicing in Parental HeLa cells. ( J ) The levels of the intronless tRNA molecules Pro and Val were measured by RT-qPCR in the samples of (I). Values have been normalized to the levels measured in Parental HeLa cells. Data values for all the presented graphs are the mean ± SEM of n≥3 independent experiments (*p

    Techniques Used: Activity Assay, Stable Transfection, Expressing, Mutagenesis, Real-time Polymerase Chain Reaction, Transfection, Quantitative RT-PCR

    17) Product Images from "Direct Inhibition of the Allergic Effector Response by Raw Cow’s Milk—An Extensive In Vitro Assessment"

    Article Title: Direct Inhibition of the Allergic Effector Response by Raw Cow’s Milk—An Extensive In Vitro Assessment

    Journal: Cells

    doi: 10.3390/cells9051258

    Membrane-bound IgE expression retained after DNP-HSA stimulation in raw milk-treated BMMC. After incubation with the different milk types and IgE-mediated activation by a range of DNP-HSA concentrations, BMMC were directly lysed in Triton X-100 for SDS-PAGE and immunoblotting. ( A ) Triton X-100 soluble fraction of BMMC lysates immunoblotted for IgE and β-actin. ( B ) Densitometric values of IgE normalized to β-actin. ( C ) Membrane-bound IgE expression relative to the control group at 6.25 ng/mL DNP-HSA. Densitometric values are representative of three independent experiments ( A – B ). Membrane-bound IgE expression at 6.25 ng/mL DNP-HSA is presented as mean ± SEM of three independent experiments ( n = 3; C ). Statistical analysis was performed compared to the raw milk group using one-way ANOVA, followed by Dunnett’s multiple comparisons test. M, marker; raw, raw cow’s milk; heated, heated raw cow’s milk; shop, shop milk; IgE-HC, IgE heavy chain; DNP-HSA, 2,4-dinitrophenol conjugated to human serum albumin.
    Figure Legend Snippet: Membrane-bound IgE expression retained after DNP-HSA stimulation in raw milk-treated BMMC. After incubation with the different milk types and IgE-mediated activation by a range of DNP-HSA concentrations, BMMC were directly lysed in Triton X-100 for SDS-PAGE and immunoblotting. ( A ) Triton X-100 soluble fraction of BMMC lysates immunoblotted for IgE and β-actin. ( B ) Densitometric values of IgE normalized to β-actin. ( C ) Membrane-bound IgE expression relative to the control group at 6.25 ng/mL DNP-HSA. Densitometric values are representative of three independent experiments ( A – B ). Membrane-bound IgE expression at 6.25 ng/mL DNP-HSA is presented as mean ± SEM of three independent experiments ( n = 3; C ). Statistical analysis was performed compared to the raw milk group using one-way ANOVA, followed by Dunnett’s multiple comparisons test. M, marker; raw, raw cow’s milk; heated, heated raw cow’s milk; shop, shop milk; IgE-HC, IgE heavy chain; DNP-HSA, 2,4-dinitrophenol conjugated to human serum albumin.

    Techniques Used: Expressing, Incubation, Activation Assay, SDS Page, Marker

    18) Product Images from "Ethanol-triggered Lipophagy Requires SQSTM1 in AML12 Hepatic Cells"

    Article Title: Ethanol-triggered Lipophagy Requires SQSTM1 in AML12 Hepatic Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12485-2

    SQSTM1 is important for LC3 to be associated with LDs and ethanol-induced lipophagy. ( A ) AML12 cells were transfected with scrambled (Scr) siRNA or SQSTM1-specific siRNA, followed by treatment with ethanol plus/minus CQ for 24 hours before being analyzed by immunoblotting and densitometry. The protein levels were normalized to that of β-actin and expressed as fold of the control level. ( B and C ) AML12 cells were transfected with scrambled (Scr) siRNA or SQSTM1-specific siRNA, treated with ethanol for 24 hours and then stained for LC3, SQSTM1 and LDs. Representative confocal images with two or three-color merge were shown ( B ). Paired co-localizations of LC3, SQSTM1 and LDs were individually illustrated for the boxed areas of the ethanol/CQ-treated samples. The percent of LDs with co-localized LC3 signals was determined ( C ). ( D and E ) AML12 cells subjected to SQSTM1 knockdown as described above were collected to determine the intracellular levels of TG ( D ) and cholesterol ( E ). Data represent mean ± SEM. * P
    Figure Legend Snippet: SQSTM1 is important for LC3 to be associated with LDs and ethanol-induced lipophagy. ( A ) AML12 cells were transfected with scrambled (Scr) siRNA or SQSTM1-specific siRNA, followed by treatment with ethanol plus/minus CQ for 24 hours before being analyzed by immunoblotting and densitometry. The protein levels were normalized to that of β-actin and expressed as fold of the control level. ( B and C ) AML12 cells were transfected with scrambled (Scr) siRNA or SQSTM1-specific siRNA, treated with ethanol for 24 hours and then stained for LC3, SQSTM1 and LDs. Representative confocal images with two or three-color merge were shown ( B ). Paired co-localizations of LC3, SQSTM1 and LDs were individually illustrated for the boxed areas of the ethanol/CQ-treated samples. The percent of LDs with co-localized LC3 signals was determined ( C ). ( D and E ) AML12 cells subjected to SQSTM1 knockdown as described above were collected to determine the intracellular levels of TG ( D ) and cholesterol ( E ). Data represent mean ± SEM. * P

    Techniques Used: Transfection, Staining

    Ethanol-induced lipid accumulation is affected by ethanol metabolism and lysosome function. ( A – D ) AML12 cells were treated with ethanol for 24 hours in the presence or absence of 4-MP ( A , B ), or CQ ( C , D ). Intracellular levels of TG ( A , C ) and cholesterol ( B,D ) were then determined. ( E ) AML12 cells were incubated with ethanol (24 h) with or without CQ, and then stained with 1 µM Bodipy-581/591 for lipid droplets (LD), which were quantified. ( F ) AML12 cells were cultured on coverslips in 24-well plates, infected with adenoviral GFP-LC3 for 24 h, and then treated with ethanol for 24 hours with or without CQ. The number of GFP-LC3 puncta were quantified. Boxed areas are enlarged in the inserts, showing the GFP-LC3 puncta. ( G , H ) Cells were cultured with ethanol for 12 hours ( G ) or 24 hours ( H ) with or without CQ. Cell lysates were examined by immunoblotting for SQSTM1 and LC3. Densitometry was conducted, normalized to β-actin and expressed as fold of the control level. In all experiments, CQ treatment was only for the last three hours of the culture. Data represent mean ± SEM. * P
    Figure Legend Snippet: Ethanol-induced lipid accumulation is affected by ethanol metabolism and lysosome function. ( A – D ) AML12 cells were treated with ethanol for 24 hours in the presence or absence of 4-MP ( A , B ), or CQ ( C , D ). Intracellular levels of TG ( A , C ) and cholesterol ( B,D ) were then determined. ( E ) AML12 cells were incubated with ethanol (24 h) with or without CQ, and then stained with 1 µM Bodipy-581/591 for lipid droplets (LD), which were quantified. ( F ) AML12 cells were cultured on coverslips in 24-well plates, infected with adenoviral GFP-LC3 for 24 h, and then treated with ethanol for 24 hours with or without CQ. The number of GFP-LC3 puncta were quantified. Boxed areas are enlarged in the inserts, showing the GFP-LC3 puncta. ( G , H ) Cells were cultured with ethanol for 12 hours ( G ) or 24 hours ( H ) with or without CQ. Cell lysates were examined by immunoblotting for SQSTM1 and LC3. Densitometry was conducted, normalized to β-actin and expressed as fold of the control level. In all experiments, CQ treatment was only for the last three hours of the culture. Data represent mean ± SEM. * P

    Techniques Used: Incubation, Staining, Cell Culture, Infection

    19) Product Images from "An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity"

    Article Title: An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

    Journal: Nature cell biology

    doi: 10.1038/ncb3592

    MRCK activates apical actomyosin contractility that controls apical morphogenesis. ( a ) Spontaneous polarization of MDCK cells leads to the formation of apical actomyosin caps positive for p-MLC and F-actin. ( b ) Expression of MRCKβ as analysed by immunoblotting of total cell extracts. ( c,d ) Scanning electron microscopy of apical domains reveals levels of microvilli induction by MDCK cells upon depletion of MRCK without or with complementation with exogenously expressed MRCKβ-flag. ( e,f ) Measurement of active Myosin at cortical caps (A) and basal membrane (B) during polarization and differentiation of cells depleted of or rescued for MRCK expression by confocal immunofluorescence microscopy. ( g,h ) Measured cell height and F-actin levels in polarizing cells with or without MRCK function. For all quantifications, n=3 independent experiments and shown are the data points, means ± 1 SD (in black), the total number of cells analysed for each type of sample across all experiments, and p-values derived from t-tests. Arrowheads point to the apical cortex demarked by F-actin. Unprocessed original scans of blots are shown in Supplementary Figure 8 . Scale bars: electron micrographs, 1μm; confocal immunofluorescence images, 10μm.
    Figure Legend Snippet: MRCK activates apical actomyosin contractility that controls apical morphogenesis. ( a ) Spontaneous polarization of MDCK cells leads to the formation of apical actomyosin caps positive for p-MLC and F-actin. ( b ) Expression of MRCKβ as analysed by immunoblotting of total cell extracts. ( c,d ) Scanning electron microscopy of apical domains reveals levels of microvilli induction by MDCK cells upon depletion of MRCK without or with complementation with exogenously expressed MRCKβ-flag. ( e,f ) Measurement of active Myosin at cortical caps (A) and basal membrane (B) during polarization and differentiation of cells depleted of or rescued for MRCK expression by confocal immunofluorescence microscopy. ( g,h ) Measured cell height and F-actin levels in polarizing cells with or without MRCK function. For all quantifications, n=3 independent experiments and shown are the data points, means ± 1 SD (in black), the total number of cells analysed for each type of sample across all experiments, and p-values derived from t-tests. Arrowheads point to the apical cortex demarked by F-actin. Unprocessed original scans of blots are shown in Supplementary Figure 8 . Scale bars: electron micrographs, 1μm; confocal immunofluorescence images, 10μm.

    Techniques Used: Expressing, Electron Microscopy, Immunofluorescence, Microscopy, Derivative Assay

    20) Product Images from "In vitro and In vivo imaging of antivasculogenesis induced by Noggin protein expression in human venous endothelial cells"

    Article Title: In vitro and In vivo imaging of antivasculogenesis induced by Noggin protein expression in human venous endothelial cells

    Journal: The FASEB Journal

    doi: 10.1096/fj.08-127795

    Analysis of Noggin expression in transduced HUVECs. A ) RT-PCR of total RNA using Noggin, BMP-2, BMP-4, BMP receptor (type IA, B) and β-actin specific primers. B ) Semiquantitation of results shown in A . C ) Immunoblotting of HUVEC lysates and concentrated conditioned media using rabbit polyclonal anti-Noggin antibody followed by anti-rabbit-peroxidase conjugate. D ) Comparative growth rates of WT, GFP + , and Nog + /GFP HUVECs. Normalized levels of Nog and BMP-4 mRNA in GFP/Nog + cells were statistically significantly different from WT and GFP + cells. * P
    Figure Legend Snippet: Analysis of Noggin expression in transduced HUVECs. A ) RT-PCR of total RNA using Noggin, BMP-2, BMP-4, BMP receptor (type IA, B) and β-actin specific primers. B ) Semiquantitation of results shown in A . C ) Immunoblotting of HUVEC lysates and concentrated conditioned media using rabbit polyclonal anti-Noggin antibody followed by anti-rabbit-peroxidase conjugate. D ) Comparative growth rates of WT, GFP + , and Nog + /GFP HUVECs. Normalized levels of Nog and BMP-4 mRNA in GFP/Nog + cells were statistically significantly different from WT and GFP + cells. * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, IA

    21) Product Images from "A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm"

    Article Title: A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm

    Journal: The Plant Cell

    doi: 10.1105/tpc.022178

    Detection of CDKA-DN Expression in Developing Transgenic Maize Endosperms. Endosperm protein extracts (40 μg/lane) from 8- to 18-DAP kernels were separated by 12.5% SDS-PAGE and blotted onto nitrocellulose. (A) Immunoblotting with monoclonal anti-HA antibody detected a 36-kD protein corresponding to CDKA-DN. (B) Immunoblotting with polyclonal anti-CDKA antibody detected CDKA-DN and a 34-kD protein corresponding to the endogenous CDKA. (C) CDKA transcripts were detected in 16-DAP endosperm by semiquantitative RT-PCR using gene-specific primers to amplify the transgene (CDKA-DN), both the endogenous and transgenic CDKA (CDKA + CDKA-DN), and actin, which was used as loading control. RT-PCR products from individual CDKA-DN endosperms are shown in the first two lanes (CDKA-DN), and those from nontransgenic controls are in the last two lanes (control).
    Figure Legend Snippet: Detection of CDKA-DN Expression in Developing Transgenic Maize Endosperms. Endosperm protein extracts (40 μg/lane) from 8- to 18-DAP kernels were separated by 12.5% SDS-PAGE and blotted onto nitrocellulose. (A) Immunoblotting with monoclonal anti-HA antibody detected a 36-kD protein corresponding to CDKA-DN. (B) Immunoblotting with polyclonal anti-CDKA antibody detected CDKA-DN and a 34-kD protein corresponding to the endogenous CDKA. (C) CDKA transcripts were detected in 16-DAP endosperm by semiquantitative RT-PCR using gene-specific primers to amplify the transgene (CDKA-DN), both the endogenous and transgenic CDKA (CDKA + CDKA-DN), and actin, which was used as loading control. RT-PCR products from individual CDKA-DN endosperms are shown in the first two lanes (CDKA-DN), and those from nontransgenic controls are in the last two lanes (control).

    Techniques Used: Expressing, Transgenic Assay, SDS Page, Reverse Transcription Polymerase Chain Reaction

    22) Product Images from "A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm"

    Article Title: A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm

    Journal: The Plant Cell

    doi: 10.1105/tpc.022178

    Detection of CDKA-DN Expression in Developing Transgenic Maize Endosperms. Endosperm protein extracts (40 μg/lane) from 8- to 18-DAP kernels were separated by 12.5% SDS-PAGE and blotted onto nitrocellulose. (A) Immunoblotting with monoclonal anti-HA antibody detected a 36-kD protein corresponding to CDKA-DN. (B) Immunoblotting with polyclonal anti-CDKA antibody detected CDKA-DN and a 34-kD protein corresponding to the endogenous CDKA. (C) CDKA transcripts were detected in 16-DAP endosperm by semiquantitative RT-PCR using gene-specific primers to amplify the transgene (CDKA-DN), both the endogenous and transgenic CDKA (CDKA + CDKA-DN), and actin, which was used as loading control. RT-PCR products from individual CDKA-DN endosperms are shown in the first two lanes (CDKA-DN), and those from nontransgenic controls are in the last two lanes (control).
    Figure Legend Snippet: Detection of CDKA-DN Expression in Developing Transgenic Maize Endosperms. Endosperm protein extracts (40 μg/lane) from 8- to 18-DAP kernels were separated by 12.5% SDS-PAGE and blotted onto nitrocellulose. (A) Immunoblotting with monoclonal anti-HA antibody detected a 36-kD protein corresponding to CDKA-DN. (B) Immunoblotting with polyclonal anti-CDKA antibody detected CDKA-DN and a 34-kD protein corresponding to the endogenous CDKA. (C) CDKA transcripts were detected in 16-DAP endosperm by semiquantitative RT-PCR using gene-specific primers to amplify the transgene (CDKA-DN), both the endogenous and transgenic CDKA (CDKA + CDKA-DN), and actin, which was used as loading control. RT-PCR products from individual CDKA-DN endosperms are shown in the first two lanes (CDKA-DN), and those from nontransgenic controls are in the last two lanes (control).

    Techniques Used: Expressing, Transgenic Assay, SDS Page, Reverse Transcription Polymerase Chain Reaction

    23) Product Images from "Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17"

    Article Title: Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2013.06.010

    The tetratricopeptide repeat-containing proteins TTC30A1, TTC30A2 and TTC30B interact with IFT52 (A) HEK293 cells were transfected with expression vectors for AU1-TTC30A1, AU1-TTC30A2 or AU1-TTC30B and FLAG tagged-IFT52 as indicated. At 20 hours after transfection, whole cell extracts were prepared. (A) Samples were analyzed by denaturing gel electrophoresis and immunoblotting with antibody to AU1 to detect the input expression of the tagged-TTC30 isoforms. A non-specific band was detected in all lanes. (B) Input expression of FLAG-IFT52 was detected by immunoblotting with FLAG monoclonal antibody. (C) To detect interaction of AU-tagged TTC30 isoforms with IFT52, FLAG-tagged proteins were isolated by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with AU1 antibody to detect co-immunoprecipitated AU1-TTC30A1, AU1-TTC30A2 or AU1-TTC30B. (D) Imunoblotting of cell extracts with antibody to ERK-1 as a loading control.
    Figure Legend Snippet: The tetratricopeptide repeat-containing proteins TTC30A1, TTC30A2 and TTC30B interact with IFT52 (A) HEK293 cells were transfected with expression vectors for AU1-TTC30A1, AU1-TTC30A2 or AU1-TTC30B and FLAG tagged-IFT52 as indicated. At 20 hours after transfection, whole cell extracts were prepared. (A) Samples were analyzed by denaturing gel electrophoresis and immunoblotting with antibody to AU1 to detect the input expression of the tagged-TTC30 isoforms. A non-specific band was detected in all lanes. (B) Input expression of FLAG-IFT52 was detected by immunoblotting with FLAG monoclonal antibody. (C) To detect interaction of AU-tagged TTC30 isoforms with IFT52, FLAG-tagged proteins were isolated by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with AU1 antibody to detect co-immunoprecipitated AU1-TTC30A1, AU1-TTC30A2 or AU1-TTC30B. (D) Imunoblotting of cell extracts with antibody to ERK-1 as a loading control.

    Techniques Used: Transfection, Expressing, Nucleic Acid Electrophoresis, Isolation, Immunoprecipitation

    Expression of tagged mouse IFT57 to isolate an IFT complex from mouse pituitary cells (A) Organization of the coding sequence of tagged mouse IFT57. The mouse IFT57 coding sequence was modified so that a calmodulin binding domain (CalB), an epitope recognized by the FLAG antibody (FLAG), 2 recognition/cleavage sites for the TEV protease (2xTEV) and 2 epitopes recognized by the AU1 antibody were added to the caroboxy-terminus of the protein. (B) Coding sequence for tagged IFT57. The first 3 amino acids and the last three amino acids of IFT57 are shown (upper case), and the sequence added to the carboxy-terminus of the protein is shown (lower case). The calmodulin binding domain, FLAG epitope, TEV recognition/cleavage sites and AU1 epitopes of the carboxy-terminal tag are indicated by underlines. (C) Expression of tagged IFT57 in the αT3-1 pituitary cell line. Cell extracts were prepared from either control αT3-1 cell or αT3-1 cells stably expressing tagged IFT57. Cell extracts were resolved by denaturing polyacrylamide gel electrophoresis, transferred to a membrane and then incubated with FLAG antiserum to detect input-tagged IFT57 or with antiserum to IFT172 to detect endogenous IFT172. To assay for formation of an IFT complex, the tagged-IFT57 was isolated by immunoprecipitation with FLAG monoclonal antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with antibody to IFT172 to detect co-immunoprecipitated IFT172. (D) Identification of proteins interacting with tagged IFT57 in pituitary cells. Whole cell extracts were prepared from control αT3-1 cells or αT3-1 cells expressing tagged IFT57. The cell extracts were incubated with AUI antibody immobilized on agarose beads. After washing of the beads, the bound proteins were eluted by digestion with TEV protease and the eluate incubated with FLAG antibodies immobilized on agarose beads. After washing, bound proteins were eluted with FLAG peptide and resolved by denaturing polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250. Stained bands that appeared to be substantially more intense in the sample from the tagged IFT57 cells were cut from the gel and subject to trypsin digestion and mass spectrometry. Several known IFT proteins were identified (labeled to the right of the gel lanes) and one 70 kDa protein. Some bands were not determined (n.d.).
    Figure Legend Snippet: Expression of tagged mouse IFT57 to isolate an IFT complex from mouse pituitary cells (A) Organization of the coding sequence of tagged mouse IFT57. The mouse IFT57 coding sequence was modified so that a calmodulin binding domain (CalB), an epitope recognized by the FLAG antibody (FLAG), 2 recognition/cleavage sites for the TEV protease (2xTEV) and 2 epitopes recognized by the AU1 antibody were added to the caroboxy-terminus of the protein. (B) Coding sequence for tagged IFT57. The first 3 amino acids and the last three amino acids of IFT57 are shown (upper case), and the sequence added to the carboxy-terminus of the protein is shown (lower case). The calmodulin binding domain, FLAG epitope, TEV recognition/cleavage sites and AU1 epitopes of the carboxy-terminal tag are indicated by underlines. (C) Expression of tagged IFT57 in the αT3-1 pituitary cell line. Cell extracts were prepared from either control αT3-1 cell or αT3-1 cells stably expressing tagged IFT57. Cell extracts were resolved by denaturing polyacrylamide gel electrophoresis, transferred to a membrane and then incubated with FLAG antiserum to detect input-tagged IFT57 or with antiserum to IFT172 to detect endogenous IFT172. To assay for formation of an IFT complex, the tagged-IFT57 was isolated by immunoprecipitation with FLAG monoclonal antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with antibody to IFT172 to detect co-immunoprecipitated IFT172. (D) Identification of proteins interacting with tagged IFT57 in pituitary cells. Whole cell extracts were prepared from control αT3-1 cells or αT3-1 cells expressing tagged IFT57. The cell extracts were incubated with AUI antibody immobilized on agarose beads. After washing of the beads, the bound proteins were eluted by digestion with TEV protease and the eluate incubated with FLAG antibodies immobilized on agarose beads. After washing, bound proteins were eluted with FLAG peptide and resolved by denaturing polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250. Stained bands that appeared to be substantially more intense in the sample from the tagged IFT57 cells were cut from the gel and subject to trypsin digestion and mass spectrometry. Several known IFT proteins were identified (labeled to the right of the gel lanes) and one 70 kDa protein. Some bands were not determined (n.d.).

    Techniques Used: Expressing, Sequencing, Modification, Binding Assay, FLAG-tag, Stable Transfection, Polyacrylamide Gel Electrophoresis, Incubation, Isolation, Immunoprecipitation, Nucleic Acid Electrophoresis, Staining, Mass Spectrometry, Labeling

    Interaction of TTC30B and KIF17 with IFT52 and IFT57 in cells (A) HEK293 cells were transfected with expression vectors for AU1-tagged TTC30B or HA-tagged KIF17 and FLAG tagged-IFT52 or FLAG-tagged IFT57 as indicated. At 20 hours after transfection, whole cell extracts were prepared. (A) Samples were analyzed by denaturing gel electrophoresis and immunoblotting with antibody to AU1 to detect the input expression of TTC30B. (B) To detect interaction of TTC30B with IFT52 or IFT57, FLAG-tagged proteins were isolated by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with AU1 antibody to detect co-immunoprecipitated AU1-TTC30B. (C) Input expression of HA-KIF17 was detected by gel electrophoresis and immunoblotting with HA antibody. (D) Interaction of KIF17 with the IFT complex was analyzed by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with HA antibody to detect co-immunoprecipitated HA-KIF17.
    Figure Legend Snippet: Interaction of TTC30B and KIF17 with IFT52 and IFT57 in cells (A) HEK293 cells were transfected with expression vectors for AU1-tagged TTC30B or HA-tagged KIF17 and FLAG tagged-IFT52 or FLAG-tagged IFT57 as indicated. At 20 hours after transfection, whole cell extracts were prepared. (A) Samples were analyzed by denaturing gel electrophoresis and immunoblotting with antibody to AU1 to detect the input expression of TTC30B. (B) To detect interaction of TTC30B with IFT52 or IFT57, FLAG-tagged proteins were isolated by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with AU1 antibody to detect co-immunoprecipitated AU1-TTC30B. (C) Input expression of HA-KIF17 was detected by gel electrophoresis and immunoblotting with HA antibody. (D) Interaction of KIF17 with the IFT complex was analyzed by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with HA antibody to detect co-immunoprecipitated HA-KIF17.

    Techniques Used: Transfection, Expressing, Nucleic Acid Electrophoresis, Isolation, Immunoprecipitation

    24) Product Images from "PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1–Parkin-mediated mitophagy"

    Article Title: PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1–Parkin-mediated mitophagy

    Journal: Cell Research

    doi: 10.1038/s41422-018-0056-0

    PTEN-L keeps Parkin in closed conformation by enhancing the interaction of Parkin UBL and RING1 domains in a protein phosphatase activity-dependent manner. a Construction of PTEN-L truncations. PTEN-L contains an ATR region, a phosphatase domain and a C-terminal region with a C2 domain and a C-Tail domain. PTEN-L-C297S is a dual lipid-protein phosphatase-defective mutant, while PTEN-L-G302R is a lipid phosphatase-defective mutant. b , c HEK293T cells transfected with GFP-Parkin and different constructs of Flag-tagged PTEN-L were treated without or with CCCP (5 µM) for 4 h. PTEN-L was immunoprecipitated with anti-Flag beads followed by immunoblotting for GFP and Flag. d Construction of Flag-tagged Parkin truncations, including Parkin-FL (full length) and truncated Parkin constructs: UBL-R0, Exon4, R1-IBR-R2, UBL-R1-IBR-R2 (deletion of R0 domain) and IBR-R2. e HEK293T cells transfected with GFP-PTEN-L and different constructs of Flag-tagged Parkin were treated without or with CCCP (5 µM) for 4 h. PTEN-L was then immunoprecipitated with anti-GFP beads followed by immunoblotting for Flag and GFP. f HEK293T cells were transfected with Flag-PTEN-L or the two Flag-PTEN-L mutants, together with Parkin truncation mutants GFP-UBL and GST-RING1 (R1) or GST empty vector (pEBG). Cells were then treated with or without CCCP (20 µM) for 4 h. RING1 was immunoprecipitated with anti-GST beads followed by immunoblotting for GFP, Flag and GST. g YFP-Parkin-HeLa cells transiently transfected with mCherry-PTEN-L or the two mCherry-PTEN-L mutants were treated with CCCP (5 µM) for 2 h. YFP-Parkin (green), mCherry (red). Scale bar, 10 µm
    Figure Legend Snippet: PTEN-L keeps Parkin in closed conformation by enhancing the interaction of Parkin UBL and RING1 domains in a protein phosphatase activity-dependent manner. a Construction of PTEN-L truncations. PTEN-L contains an ATR region, a phosphatase domain and a C-terminal region with a C2 domain and a C-Tail domain. PTEN-L-C297S is a dual lipid-protein phosphatase-defective mutant, while PTEN-L-G302R is a lipid phosphatase-defective mutant. b , c HEK293T cells transfected with GFP-Parkin and different constructs of Flag-tagged PTEN-L were treated without or with CCCP (5 µM) for 4 h. PTEN-L was immunoprecipitated with anti-Flag beads followed by immunoblotting for GFP and Flag. d Construction of Flag-tagged Parkin truncations, including Parkin-FL (full length) and truncated Parkin constructs: UBL-R0, Exon4, R1-IBR-R2, UBL-R1-IBR-R2 (deletion of R0 domain) and IBR-R2. e HEK293T cells transfected with GFP-PTEN-L and different constructs of Flag-tagged Parkin were treated without or with CCCP (5 µM) for 4 h. PTEN-L was then immunoprecipitated with anti-GFP beads followed by immunoblotting for Flag and GFP. f HEK293T cells were transfected with Flag-PTEN-L or the two Flag-PTEN-L mutants, together with Parkin truncation mutants GFP-UBL and GST-RING1 (R1) or GST empty vector (pEBG). Cells were then treated with or without CCCP (20 µM) for 4 h. RING1 was immunoprecipitated with anti-GST beads followed by immunoblotting for GFP, Flag and GST. g YFP-Parkin-HeLa cells transiently transfected with mCherry-PTEN-L or the two mCherry-PTEN-L mutants were treated with CCCP (5 µM) for 2 h. YFP-Parkin (green), mCherry (red). Scale bar, 10 µm

    Techniques Used: Activity Assay, Mutagenesis, Transfection, Construct, Immunoprecipitation, Plasmid Preparation

    PTEN-L dephosphorylates ubiquitin. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 3 h. Whole-cell lysates were analyzed by immunoblotting. b YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 3 h. Immunofluorescence staining against pSer65-Ub was performed and observed by fluorescent microscopy. pSer65-Ub (red), YFP-Parkin (green), Nucleus (DAPI, blue). Scale bar, 10 µm. c YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with O/A (25 nM and 250 nM) for indicated hours and immunoblotting was performed. d Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with O/A (25 nM and 250 nM) for indicated hours and immunoblotting was performed. e In vitro dephosphorylation assay. Purified pSer65-Ub was incubated with purified Flag-PTEN-L, Flag-PTEN-L-C297S or Flag-PTEN-L-G302R in phosphatase reaction buffer and ubiquitin phosphorylation level was evaluated by immunoblotting
    Figure Legend Snippet: PTEN-L dephosphorylates ubiquitin. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 3 h. Whole-cell lysates were analyzed by immunoblotting. b YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 3 h. Immunofluorescence staining against pSer65-Ub was performed and observed by fluorescent microscopy. pSer65-Ub (red), YFP-Parkin (green), Nucleus (DAPI, blue). Scale bar, 10 µm. c YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with O/A (25 nM and 250 nM) for indicated hours and immunoblotting was performed. d Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with O/A (25 nM and 250 nM) for indicated hours and immunoblotting was performed. e In vitro dephosphorylation assay. Purified pSer65-Ub was incubated with purified Flag-PTEN-L, Flag-PTEN-L-C297S or Flag-PTEN-L-G302R in phosphatase reaction buffer and ubiquitin phosphorylation level was evaluated by immunoblotting

    Techniques Used: Expressing, Plasmid Preparation, Immunofluorescence, Staining, Microscopy, In Vitro, De-Phosphorylation Assay, Purification, Incubation

    PTEN-L disrupts the feedforward mechanism in mitophagy by targeting the pSer65-Ub chains. a In vitro dephosphorylation assay using purified pSer65-tetra-Ub. Purified Flag-PTEN-L was incubated with pSer65-tetra-Ub in the phosphatase reaction buffer for 1 h at 30 °C. Calf intestinal phosphatase (CIP) was used as a positive control. b In vitro dephosphorylation assay using purified pSer65-poly-Ub chains, following the same procedure in a . λPP was used as a positive control. c YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (10 µM) for 4 h. YFP-Parkin was pulled down by GFP beads and subjected to immunoblotting. d The MS/MS spectra of the ubiquitin peptide containing phospho-Ser65. YFP-Parkin-HeLa cells were treated with CCCP (10 µM) for 4 h and YFP-Parkin was pulled down with GFP beads. e pSer65-Ub was quantified using MS-based relative quantification analysis in YFP-Parkin-HeLa cells with or without PTEN-L stable expression after CCCP (10 µM) and O/A (25 nM and 250 nM) treatment. Data are presented as mean ± SD from 3 independent experiments. *** P
    Figure Legend Snippet: PTEN-L disrupts the feedforward mechanism in mitophagy by targeting the pSer65-Ub chains. a In vitro dephosphorylation assay using purified pSer65-tetra-Ub. Purified Flag-PTEN-L was incubated with pSer65-tetra-Ub in the phosphatase reaction buffer for 1 h at 30 °C. Calf intestinal phosphatase (CIP) was used as a positive control. b In vitro dephosphorylation assay using purified pSer65-poly-Ub chains, following the same procedure in a . λPP was used as a positive control. c YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (10 µM) for 4 h. YFP-Parkin was pulled down by GFP beads and subjected to immunoblotting. d The MS/MS spectra of the ubiquitin peptide containing phospho-Ser65. YFP-Parkin-HeLa cells were treated with CCCP (10 µM) for 4 h and YFP-Parkin was pulled down with GFP beads. e pSer65-Ub was quantified using MS-based relative quantification analysis in YFP-Parkin-HeLa cells with or without PTEN-L stable expression after CCCP (10 µM) and O/A (25 nM and 250 nM) treatment. Data are presented as mean ± SD from 3 independent experiments. *** P

    Techniques Used: In Vitro, De-Phosphorylation Assay, Purification, Incubation, Positive Control, Expressing, Plasmid Preparation, Mass Spectrometry

    PTEN-L negatively regulates mitophagy induced by various mitochondria-damaging agents. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 24 h and immunoblotting for mitochondrial proteins was performed as indicated. b Quantification of mitochondrial proteins from a . c Degradation of mtDNA. Representative images of YFP-Parkin-HeLa cells with PTEN-L stable expression and control vector immunostained to label mtDNA (red) after treatment with CCCP (5 µM) for 24 h. d Quantification of mtDNA from > 300 cells per group. Scale bar, 10 µm. e Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with CCCP (4 µM) for 24 h and immunoblotting was performed as indicated. f Quantification of data from e . Data in b , d , f are presented as mean ± SD from three independent experiments. *** P
    Figure Legend Snippet: PTEN-L negatively regulates mitophagy induced by various mitochondria-damaging agents. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 24 h and immunoblotting for mitochondrial proteins was performed as indicated. b Quantification of mitochondrial proteins from a . c Degradation of mtDNA. Representative images of YFP-Parkin-HeLa cells with PTEN-L stable expression and control vector immunostained to label mtDNA (red) after treatment with CCCP (5 µM) for 24 h. d Quantification of mtDNA from > 300 cells per group. Scale bar, 10 µm. e Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with CCCP (4 µM) for 24 h and immunoblotting was performed as indicated. f Quantification of data from e . Data in b , d , f are presented as mean ± SD from three independent experiments. *** P

    Techniques Used: Expressing, Plasmid Preparation

    PTEN-L impairs Parkin E3 ligase activity and reduces pSer65-Parkin. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for indicated hours. YFP-Parkin was immunoprecipitated with anti-GFP antibody. Immunoprecipitants (IPs) and whole-cell lysates (WCLs) were analyzed for YFP-Parkin, mitofusin-2 (MFN2), Tom20, PTEN-L, PTEN and tubulin. b YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with O/A (10 nM and 100 nM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. c Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with CCCP (5 µM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. d Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with O/A (10 nM and 100 nM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. e The MS/MS spectra of the Parkin peptide containing phospho-Ser65. YFP-Parkin-HeLa cells were treated with CCCP (10 µM) for 4 h. YFP-Parkin was pulled down with GFP beads and subjected to MS/MS analysis. f pSer65-Parkin was quantified using MS-based relative quantification analysis in YFP-Parkin-HeLa cells with or without PTEN-L stable expression after CCCP (10 µM) and O/A (25 nM and 250 nM) treatment. Data are presented as mean ± SD from 3 independent experiments. ** P
    Figure Legend Snippet: PTEN-L impairs Parkin E3 ligase activity and reduces pSer65-Parkin. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for indicated hours. YFP-Parkin was immunoprecipitated with anti-GFP antibody. Immunoprecipitants (IPs) and whole-cell lysates (WCLs) were analyzed for YFP-Parkin, mitofusin-2 (MFN2), Tom20, PTEN-L, PTEN and tubulin. b YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with O/A (10 nM and 100 nM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. c Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with CCCP (5 µM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. d Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with O/A (10 nM and 100 nM) for indicated hours and the WCLs were analyzed by immunoblotting as indicated. e The MS/MS spectra of the Parkin peptide containing phospho-Ser65. YFP-Parkin-HeLa cells were treated with CCCP (10 µM) for 4 h. YFP-Parkin was pulled down with GFP beads and subjected to MS/MS analysis. f pSer65-Parkin was quantified using MS-based relative quantification analysis in YFP-Parkin-HeLa cells with or without PTEN-L stable expression after CCCP (10 µM) and O/A (25 nM and 250 nM) treatment. Data are presented as mean ± SD from 3 independent experiments. ** P

    Techniques Used: Activity Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry

    25) Product Images from "The Bacillus subtilis yaaH Gene Is Transcribed by SigE RNA Polymerase during Sporulation, and Its Product Is Involved in Germination of Spores"

    Article Title: The Bacillus subtilis yaaH Gene Is Transcribed by SigE RNA Polymerase during Sporulation, and Its Product Is Involved in Germination of Spores

    Journal: Journal of Bacteriology

    doi:

    Detection of YaaH-His in mature spores. Spore proteins were solubilized from B. subtilis 168 transformants carrying control vector pTUBE1200H6 (lane 1) or pYAAH8 (lane 2) as described in Materials and Methods. The proteins were resolved by SDS-PAGE (15% acrylamide gel) and visualized by Coomassie brilliant blue staining (A) or immunoblotting with anti-His tag antiserum (B). Arrowheads indicate the deduced molecular mass of the YaaH-His protein.
    Figure Legend Snippet: Detection of YaaH-His in mature spores. Spore proteins were solubilized from B. subtilis 168 transformants carrying control vector pTUBE1200H6 (lane 1) or pYAAH8 (lane 2) as described in Materials and Methods. The proteins were resolved by SDS-PAGE (15% acrylamide gel) and visualized by Coomassie brilliant blue staining (A) or immunoblotting with anti-His tag antiserum (B). Arrowheads indicate the deduced molecular mass of the YaaH-His protein.

    Techniques Used: Plasmid Preparation, SDS Page, Acrylamide Gel Assay, Staining

    Detection of YaaH-His in sporulating cells. (A) pYAAH8 has replication origins available in both E. coli ( ori-E ) and B. subtilis ( ori-B ) cells, and it confers tetracycline resistance on these organisms. The yaaH gene in pYAAH8 is regulated by a promoter located upstream of the gene and fused to a sequence encoding six consecutive histidine residues (His tag). (B) B. subtilis 168 was transformed with control vector pTUBE1200H6 or pYAAH8 as described in Materials and Methods. Proteins prepared from the transformants were resolved by SDS-PAGE (15% acrylamide gel) and visualized by immunoblotting with antisera against the His tag and SigK. Arrowheads indicate the positions of the YaaH-His protein and SigK. T, harvesting times for cells (in hours).
    Figure Legend Snippet: Detection of YaaH-His in sporulating cells. (A) pYAAH8 has replication origins available in both E. coli ( ori-E ) and B. subtilis ( ori-B ) cells, and it confers tetracycline resistance on these organisms. The yaaH gene in pYAAH8 is regulated by a promoter located upstream of the gene and fused to a sequence encoding six consecutive histidine residues (His tag). (B) B. subtilis 168 was transformed with control vector pTUBE1200H6 or pYAAH8 as described in Materials and Methods. Proteins prepared from the transformants were resolved by SDS-PAGE (15% acrylamide gel) and visualized by immunoblotting with antisera against the His tag and SigK. Arrowheads indicate the positions of the YaaH-His protein and SigK. T, harvesting times for cells (in hours).

    Techniques Used: Sequencing, Transformation Assay, Plasmid Preparation, SDS Page, Acrylamide Gel Assay

    26) Product Images from "Anti‐inflammatory roles of p38α MAPK in macrophages are context dependent and require IL‐10"

    Article Title: Anti‐inflammatory roles of p38α MAPK in macrophages are context dependent and require IL‐10

    Journal: Journal of Leukocyte Biology

    doi: 10.1189/jlb.2AB0116-009RR

    p38α in macrophages regulates production of proinflammatory cytokines in a stimulus‐ and time‐dependent manner. (A) RNA was isolated from naïve BMDM from male WT and p38αCKO (CKO) mice ( n = 3), and Mapk14 mRNA abundance was assayed using quantitative RT‐PCR, with the housekeeping gene B2m as a normalizer, and then normalized further to the average expression in WT as equal to 1. BMDMs were stimulated with 100 ng/ml LPS or 50 µg/ml MTB for the indicated lengths of time (minutes). (B) Cells were lysed at the indicated time points (minutes) and subjected to immunoblotting for phosphorylated p38 (p‐p38), total p38α, and GAPDH (as a loading control). (C–F) The production of the TNF‐α (C and D) and IL‐6 (E and F) at the indicated time points (4 and 24 h) in the cell supernatants was quantified by ELISA. Significance of differences was determined by t ‐test (A) and 2‐way ANOVA, followed by Sidak's post hoc comparison tests for individual time points, comparing WT with CKO. Symbols indicate a significant difference between WT and CKO, as follows: * P
    Figure Legend Snippet: p38α in macrophages regulates production of proinflammatory cytokines in a stimulus‐ and time‐dependent manner. (A) RNA was isolated from naïve BMDM from male WT and p38αCKO (CKO) mice ( n = 3), and Mapk14 mRNA abundance was assayed using quantitative RT‐PCR, with the housekeeping gene B2m as a normalizer, and then normalized further to the average expression in WT as equal to 1. BMDMs were stimulated with 100 ng/ml LPS or 50 µg/ml MTB for the indicated lengths of time (minutes). (B) Cells were lysed at the indicated time points (minutes) and subjected to immunoblotting for phosphorylated p38 (p‐p38), total p38α, and GAPDH (as a loading control). (C–F) The production of the TNF‐α (C and D) and IL‐6 (E and F) at the indicated time points (4 and 24 h) in the cell supernatants was quantified by ELISA. Significance of differences was determined by t ‐test (A) and 2‐way ANOVA, followed by Sidak's post hoc comparison tests for individual time points, comparing WT with CKO. Symbols indicate a significant difference between WT and CKO, as follows: * P

    Techniques Used: Isolation, Mouse Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

    27) Product Images from "Paramyxovirus V Protein Interaction with the Antiviral Sensor LGP2 Disrupts MDA5 Signaling Enhancement but Is Not Relevant to LGP2-Mediated RLR Signaling Inhibition"

    Article Title: Paramyxovirus V Protein Interaction with the Antiviral Sensor LGP2 Disrupts MDA5 Signaling Enhancement but Is Not Relevant to LGP2-Mediated RLR Signaling Inhibition

    Journal: Journal of Virology

    doi: 10.1128/JVI.00737-14

    ). Expanded sequence alignment illustrates relevant region of all three RLR proteins within the MVBR, and residues targeted are depicted with an asterisk. Arrow points to LGP2 R455, which is R806 of MDA5 and L714 of RIG-I; closed circle indicates RIG-I L714. (B) LGP2 interactions with V proteins. FLAG-tagged LGP2 or variants were coexpressed with HA-tagged V proteins from measles, PIV5, and Nipah virus in HEK293T cells. The cell lysates were subjected to FLAG immunoaffinity purification, and detection of coprecipitation was carried out by anti-HA immunoblotting. (C) LGP2 RGL-LEY is defective for interaction with PIV5 V protein in infected cells. HEK293T cells were transfected with FLAG-tagged LGP2 or mutant and infected with PIV5 at the indicated multiplicity of infection (MOI) for 24 h. The cell lysates were subjected to FLAG immunoaffinity purification, and detection of coprecipitation was carried out by immunoblotting with antiserum that recognizes P and V proteins. (D) LGP2 RGL-LEY retains biological activity. A total of 25 ng MDA5 was expressed with WT or mutant LGP2 titrated at 4, 20, 100, or 500 ng of vector. Cells were stimulated by transfection of high-molecular-weight (HMW) poly(I·C) for 6 h prior to luciferase assays. Student's t test indicated as follows: N.S. (not significant), P > 0.015; *, P
    Figure Legend Snippet: ). Expanded sequence alignment illustrates relevant region of all three RLR proteins within the MVBR, and residues targeted are depicted with an asterisk. Arrow points to LGP2 R455, which is R806 of MDA5 and L714 of RIG-I; closed circle indicates RIG-I L714. (B) LGP2 interactions with V proteins. FLAG-tagged LGP2 or variants were coexpressed with HA-tagged V proteins from measles, PIV5, and Nipah virus in HEK293T cells. The cell lysates were subjected to FLAG immunoaffinity purification, and detection of coprecipitation was carried out by anti-HA immunoblotting. (C) LGP2 RGL-LEY is defective for interaction with PIV5 V protein in infected cells. HEK293T cells were transfected with FLAG-tagged LGP2 or mutant and infected with PIV5 at the indicated multiplicity of infection (MOI) for 24 h. The cell lysates were subjected to FLAG immunoaffinity purification, and detection of coprecipitation was carried out by immunoblotting with antiserum that recognizes P and V proteins. (D) LGP2 RGL-LEY retains biological activity. A total of 25 ng MDA5 was expressed with WT or mutant LGP2 titrated at 4, 20, 100, or 500 ng of vector. Cells were stimulated by transfection of high-molecular-weight (HMW) poly(I·C) for 6 h prior to luciferase assays. Student's t test indicated as follows: N.S. (not significant), P > 0.015; *, P

    Techniques Used: Sequencing, Immunoaffinity Purification, Infection, Transfection, Mutagenesis, Activity Assay, Plasmid Preparation, Molecular Weight, Luciferase

    28) Product Images from "Gp120 in the pathogenesis of human HIV-associated pain"

    Article Title: Gp120 in the pathogenesis of human HIV-associated pain

    Journal: Annals of neurology

    doi: 10.1002/ana.24139

    Comparison of HIV-1 viral loads in the ‘pain-positive’ and ‘pain-negative’ HIV-infected patients. A. HIV-1 viral loads as measured by the copy number of gag/pol RNA. Shown are the copy numbers of individual patients. B. Immunoblotting of P24 protein in the SDH in ‘pain-positive’ and ‘pain-negative’ HIV-infected patients. C. Quantitative summary of B. Error bars: SEM (#, p > 0.05, n=5/group, student's t test).
    Figure Legend Snippet: Comparison of HIV-1 viral loads in the ‘pain-positive’ and ‘pain-negative’ HIV-infected patients. A. HIV-1 viral loads as measured by the copy number of gag/pol RNA. Shown are the copy numbers of individual patients. B. Immunoblotting of P24 protein in the SDH in ‘pain-positive’ and ‘pain-negative’ HIV-infected patients. C. Quantitative summary of B. Error bars: SEM (#, p > 0.05, n=5/group, student's t test).

    Techniques Used: Infection

    29) Product Images from "Differential apoptotic response of MC3T3-E1 pre-osteoblasts to biodegradable magnesium alloys in an in vitro direct culture model"

    Article Title: Differential apoptotic response of MC3T3-E1 pre-osteoblasts to biodegradable magnesium alloys in an in vitro direct culture model

    Journal: Journal of Materials Science. Materials in Medicine

    doi: 10.1007/s10856-017-5969-5

    Representative Immunoblotting of the expression level of PARP-1, cl.PARP-1 and active casp-3 after 2, 6 and 12 days of MC3T3-E1 culture on 48-well tissue culture plates, Pure Mg, Mg2Ag and XHP Mg alloy samples a or at day 0, 2, 6 and 12 in osteogenic condition in 48 well tissue culture plates b . B-actin was used as a loading control
    Figure Legend Snippet: Representative Immunoblotting of the expression level of PARP-1, cl.PARP-1 and active casp-3 after 2, 6 and 12 days of MC3T3-E1 culture on 48-well tissue culture plates, Pure Mg, Mg2Ag and XHP Mg alloy samples a or at day 0, 2, 6 and 12 in osteogenic condition in 48 well tissue culture plates b . B-actin was used as a loading control

    Techniques Used: Expressing

    30) Product Images from "Structural Basis for Apoptosis Inhibition by Epstein-Barr Virus BHRF1"

    Article Title: Structural Basis for Apoptosis Inhibition by Epstein-Barr Virus BHRF1

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001236

    BHRF1 inhibits loss of mitochondrial transmembrane potential and Bax/Bak activation. (A) FDC-P1 cells expressing BHRF1, Bcl-2 or a control vector were treated with 25 nM staurosporine for 24 h. Outer mitochondrial transmembrane potential (Δψ m ) was assessed by DiOC 6 (3) uptake. (B) HeLa cells treated with 200 J/m 2 UV-irradiation were analyzed at 4 h for Bax translocation and cytochrome c release by immunoblotting after fractionation into soluble (s) and pellet (p) fractions. (C) Bax and Bak activation in UV-irradiated HeLa cells were assessed at 4 h using the conformation-specific mouse anti-Bak clone Ab-1 (Calbiochem) or mouse anti-Bax clone 3 antibodies [53] .
    Figure Legend Snippet: BHRF1 inhibits loss of mitochondrial transmembrane potential and Bax/Bak activation. (A) FDC-P1 cells expressing BHRF1, Bcl-2 or a control vector were treated with 25 nM staurosporine for 24 h. Outer mitochondrial transmembrane potential (Δψ m ) was assessed by DiOC 6 (3) uptake. (B) HeLa cells treated with 200 J/m 2 UV-irradiation were analyzed at 4 h for Bax translocation and cytochrome c release by immunoblotting after fractionation into soluble (s) and pellet (p) fractions. (C) Bax and Bak activation in UV-irradiated HeLa cells were assessed at 4 h using the conformation-specific mouse anti-Bak clone Ab-1 (Calbiochem) or mouse anti-Bax clone 3 antibodies [53] .

    Techniques Used: Activation Assay, Expressing, Plasmid Preparation, Irradiation, Translocation Assay, Fractionation

    31) Product Images from "Genotypic and Phenotypic Characterization of the O-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates"

    Article Title: Genotypic and Phenotypic Characterization of the O-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00794-17

    Protein glycosylation in ST-192 isolates. Data represent the reactivity of endogenous glycoproteins following immunoblotting with the glycan-specific monoclonal antibodies npg1 (specific for diNAcBAc) (A), npg2 (specific for diNAcBAc-Gal) (B), and npg3 (specific for both diNAcBac and GATDH trisaccharides) (C) and pDAb2 polyclonal antibody (specific for diNAcBac-Glc) (D). The strains used were as follows: lane 1, KS104 ( pglC ; negative control with no glycan synthesized); lane 2, positive glycan controls, including KS141 (N400 pglA ) (A), KS100 (N400) (B), KS142 (N400 pglE on ) (C), and KS966 ( pglA pglI lct :: pglH2 SK - 03-1035 ) (D); lanes 3 to 18, N. meningitidis ST-192 isolates from time points A and B as indicated. The pglA , pglE , pglH , and pglI genotype configurations for each isolate are shown below the immunoblots.
    Figure Legend Snippet: Protein glycosylation in ST-192 isolates. Data represent the reactivity of endogenous glycoproteins following immunoblotting with the glycan-specific monoclonal antibodies npg1 (specific for diNAcBAc) (A), npg2 (specific for diNAcBAc-Gal) (B), and npg3 (specific for both diNAcBac and GATDH trisaccharides) (C) and pDAb2 polyclonal antibody (specific for diNAcBac-Glc) (D). The strains used were as follows: lane 1, KS104 ( pglC ; negative control with no glycan synthesized); lane 2, positive glycan controls, including KS141 (N400 pglA ) (A), KS100 (N400) (B), KS142 (N400 pglE on ) (C), and KS966 ( pglA pglI lct :: pglH2 SK - 03-1035 ) (D); lanes 3 to 18, N. meningitidis ST-192 isolates from time points A and B as indicated. The pglA , pglE , pglH , and pglI genotype configurations for each isolate are shown below the immunoblots.

    Techniques Used: Gas Chromatography, Negative Control, Synthesized, Western Blot

    32) Product Images from "Trimethylation of Histone H3 Lysine 36 by Human Methyltransferase PRDM9 Protein *"

    Article Title: Trimethylation of Histone H3 Lysine 36 by Human Methyltransferase PRDM9 Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.523183

    Trimethylation of H3K4 and H3K36 by PRDM9 in cells. Trimethylation of H3K36 ( a ) and H3K4 ( b ) in HEK293 cells transfected with control and PRDM9 plasmids was monitored by Western immunoblotting. c , blot quantifications indicate significant H3K4 and H3K36 trimethylation in samples from cells overexpressing PRDM9. Methylation levels of Lys-4 and Lys-36 were quantified and normalized to their respective total H3 levels. FLAG-PRDM9-transfected samples were normalized to samples transfected with FLAG Control vector. IB , immunoblot; CTL , control.
    Figure Legend Snippet: Trimethylation of H3K4 and H3K36 by PRDM9 in cells. Trimethylation of H3K36 ( a ) and H3K4 ( b ) in HEK293 cells transfected with control and PRDM9 plasmids was monitored by Western immunoblotting. c , blot quantifications indicate significant H3K4 and H3K36 trimethylation in samples from cells overexpressing PRDM9. Methylation levels of Lys-4 and Lys-36 were quantified and normalized to their respective total H3 levels. FLAG-PRDM9-transfected samples were normalized to samples transfected with FLAG Control vector. IB , immunoblot; CTL , control.

    Techniques Used: Transfection, Western Blot, Methylation, Plasmid Preparation, CTL Assay

    33) Product Images from "A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets"

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    Journal: Science Advances

    doi: 10.1126/sciadv.1601470

    Rab10 acts downstream of Rab7 to facilitate the autophagic degradation of LDs. ( A and B ) Immunoblot of GST-EHBP1– or GST-RILP–mediated pulldowns for Rab7 (A) or Rab10 (B) in Hep3B hepatoma cells. Rab7 exhibits a specific affinity for RILP, whereas Rab10 interacts with both EHBP1 and RILP. ( C ) Representative immunoblotting of the results for GST or GST-RILP pulldowns of Rab7 in WT or Rab10 KO MEFs, showing that Rab10 KO does not affect the binding of Rab7 to RILP. ( D ) Immunoblotting of subcellular density gradient fractions of Hep3B cells following serum starvation in HBSS for 2 hours, followed by flotation of a CLF through an 8 to 27% discontinuous OptiPrep gradient. Boxes indicate distinct peak density fractions for either Rab10 (blue box, fraction 2) or Rab7 (red box, fraction 4). ( E ) Live-cell confocal fluorescence microscopy of a HuH-7 hepatoma cell cotransfected with both GFP-Rab7 and mCherry-Rab10 and subjected to serum starvation. LDs are stained with MDH (blue). Arrows indicate the extension of a Rab7-positive membrane away from the LD and the subsequent recruitment of a Rab10-positive structure (arrowheads) to the LD. ( F ) Quantification of the average percentage of LDs positive for the presence of Rab10 alone, Rab7 alone, or both Rab10 and Rab7 from n = 10 cells. ( G ) Quantification of the average number of Rab10-positive LDs in resting or serum-starved HuH-7 hepatoma cells treated with NT siRNA or Rab7 siRNA. ( H ) Quantification of the average number of Rab7-positive LDs in resting or serum-starved HuH-7 cells treated with NT siRNA or Rab10 siRNA. Data represent the means from n = 3 independent experiments, measuring > 50 cells per condition per repeat. *** P ≤ 0.001. Scale bars, 1 μm.
    Figure Legend Snippet: Rab10 acts downstream of Rab7 to facilitate the autophagic degradation of LDs. ( A and B ) Immunoblot of GST-EHBP1– or GST-RILP–mediated pulldowns for Rab7 (A) or Rab10 (B) in Hep3B hepatoma cells. Rab7 exhibits a specific affinity for RILP, whereas Rab10 interacts with both EHBP1 and RILP. ( C ) Representative immunoblotting of the results for GST or GST-RILP pulldowns of Rab7 in WT or Rab10 KO MEFs, showing that Rab10 KO does not affect the binding of Rab7 to RILP. ( D ) Immunoblotting of subcellular density gradient fractions of Hep3B cells following serum starvation in HBSS for 2 hours, followed by flotation of a CLF through an 8 to 27% discontinuous OptiPrep gradient. Boxes indicate distinct peak density fractions for either Rab10 (blue box, fraction 2) or Rab7 (red box, fraction 4). ( E ) Live-cell confocal fluorescence microscopy of a HuH-7 hepatoma cell cotransfected with both GFP-Rab7 and mCherry-Rab10 and subjected to serum starvation. LDs are stained with MDH (blue). Arrows indicate the extension of a Rab7-positive membrane away from the LD and the subsequent recruitment of a Rab10-positive structure (arrowheads) to the LD. ( F ) Quantification of the average percentage of LDs positive for the presence of Rab10 alone, Rab7 alone, or both Rab10 and Rab7 from n = 10 cells. ( G ) Quantification of the average number of Rab10-positive LDs in resting or serum-starved HuH-7 hepatoma cells treated with NT siRNA or Rab7 siRNA. ( H ) Quantification of the average number of Rab7-positive LDs in resting or serum-starved HuH-7 cells treated with NT siRNA or Rab10 siRNA. Data represent the means from n = 3 independent experiments, measuring > 50 cells per condition per repeat. *** P ≤ 0.001. Scale bars, 1 μm.

    Techniques Used: Binding Assay, Fluorescence, Microscopy, Staining

    34) Product Images from "Single nucleotide polymorphisms in toll-like receptor 6 are associated with altered lipopeptide- and mycobacteria-induced IL-6 secretion"

    Article Title: Single nucleotide polymorphisms in toll-like receptor 6 are associated with altered lipopeptide- and mycobacteria-induced IL-6 secretion

    Journal: Genes and immunity

    doi: 10.1038/gene.2010.14

    Regulation of NF-κB signaling by a TLR6 polymorphism HEK293 cells were transfected with an NF-κB luciferase reporter, a Renilla luciferase construct to control for transfection efficiency (pRL-TK), and CD14. Additional transfectants varied by condition and included an empty plasmid vector (EV), or, TLR2 with one of 3 TLR6 constructs, 745C, 745T, or 2039A. Polymorphism C2039A (P680H) is a TLR6 variant with a dominant–negative effect on NF-κB signaling. Luciferase activity shown represents the ratio between basal activity (medium only) and that of transfected cells stimulated with (A) PAM2, (B) LPS, or (C) Mtb Lysate. Mean values (+/− standard deviation) are depicted for three independent experiments, each performed in triplicate. P values calculated with Student’s t-test. RLU, relative luciferase units. (D) Expression of TLR6 745C (wild type) and 745T (variant) by immunoblotting using transfected but unstimulated HEK293 cells.
    Figure Legend Snippet: Regulation of NF-κB signaling by a TLR6 polymorphism HEK293 cells were transfected with an NF-κB luciferase reporter, a Renilla luciferase construct to control for transfection efficiency (pRL-TK), and CD14. Additional transfectants varied by condition and included an empty plasmid vector (EV), or, TLR2 with one of 3 TLR6 constructs, 745C, 745T, or 2039A. Polymorphism C2039A (P680H) is a TLR6 variant with a dominant–negative effect on NF-κB signaling. Luciferase activity shown represents the ratio between basal activity (medium only) and that of transfected cells stimulated with (A) PAM2, (B) LPS, or (C) Mtb Lysate. Mean values (+/− standard deviation) are depicted for three independent experiments, each performed in triplicate. P values calculated with Student’s t-test. RLU, relative luciferase units. (D) Expression of TLR6 745C (wild type) and 745T (variant) by immunoblotting using transfected but unstimulated HEK293 cells.

    Techniques Used: Transfection, Luciferase, Construct, Plasmid Preparation, Variant Assay, Dominant Negative Mutation, Activity Assay, Standard Deviation, Expressing

    35) Product Images from "Plastid Translation Elongation Factor Tu Is Prone to Heat-Induced Aggregation Despite Its Critical Role in Plant Heat Tolerance 1Plastid Translation Elongation Factor Tu Is Prone to Heat-Induced Aggregation Despite Its Critical Role in Plant Heat Tolerance 1 [OPEN]"

    Article Title: Plastid Translation Elongation Factor Tu Is Prone to Heat-Induced Aggregation Despite Its Critical Role in Plant Heat Tolerance 1Plastid Translation Elongation Factor Tu Is Prone to Heat-Induced Aggregation Despite Its Critical Role in Plant Heat Tolerance 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.17.01672

    Heat-induced rapid aggregation of Arabidopsis plastid EF-Tu in vitro. Total proteins (T) were extracted from transgenic AtRabe1b-OE , AtRabe1b-GTP-OE , and AtRabe1b-GDP-OE Arabidopsis plants and were incubated at the indicated temperatures for the indicated hours. Soluble proteins in the supernatants (S) and insoluble proteins in the pellets (P) were separated by low-speed centrifugation, and the relative amounts of myc-tagged AtRabe1b, AtRabe1b-GTP, and AtRabe1b-GDP proteins in the protein fractions were analyzed by immunoblotting using an anti-myc monoclonal antibody. Rubisco large subunit proteins stained with Ponceau S are shown as the loading control.
    Figure Legend Snippet: Heat-induced rapid aggregation of Arabidopsis plastid EF-Tu in vitro. Total proteins (T) were extracted from transgenic AtRabe1b-OE , AtRabe1b-GTP-OE , and AtRabe1b-GDP-OE Arabidopsis plants and were incubated at the indicated temperatures for the indicated hours. Soluble proteins in the supernatants (S) and insoluble proteins in the pellets (P) were separated by low-speed centrifugation, and the relative amounts of myc-tagged AtRabe1b, AtRabe1b-GTP, and AtRabe1b-GDP proteins in the protein fractions were analyzed by immunoblotting using an anti-myc monoclonal antibody. Rubisco large subunit proteins stained with Ponceau S are shown as the loading control.

    Techniques Used: In Vitro, Transgenic Assay, Incubation, Centrifugation, Staining

    Heat-induced rapid aggregation of Arabidopsis plastid EF-Tu in vivo. Transgenic AtRabe1b-OE , AtRabe1b-GTP-OE , and AtRabe1b-GDP-OE Arabidopsis plants were treated at the indicated temperatures for the indicated hours. Total proteins (T) were extracted from treated transgenic plants and separated by low-speed centrifugation into soluble proteins in the supernatants (S) and insoluble proteins in the pellets (P). Myc-tagged AtRabe1b, AtRabe1b-GTP, and AtRabe1b-GDP proteins in the total, soluble, and insoluble fractions were analyzed by immunoblotting. Rubisco large subunit proteins stained with Ponceau S are shown as the loading control.
    Figure Legend Snippet: Heat-induced rapid aggregation of Arabidopsis plastid EF-Tu in vivo. Transgenic AtRabe1b-OE , AtRabe1b-GTP-OE , and AtRabe1b-GDP-OE Arabidopsis plants were treated at the indicated temperatures for the indicated hours. Total proteins (T) were extracted from treated transgenic plants and separated by low-speed centrifugation into soluble proteins in the supernatants (S) and insoluble proteins in the pellets (P). Myc-tagged AtRabe1b, AtRabe1b-GTP, and AtRabe1b-GDP proteins in the total, soluble, and insoluble fractions were analyzed by immunoblotting. Rubisco large subunit proteins stained with Ponceau S are shown as the loading control.

    Techniques Used: In Vivo, Transgenic Assay, Centrifugation, Staining

    36) Product Images from "Phosphorylation and Ubiquitination Regulate Protein Phosphatase 5 Activity and Its Prosurvival Role in Kidney Cancer"

    Article Title: Phosphorylation and Ubiquitination Regulate Protein Phosphatase 5 Activity and Its Prosurvival Role in Kidney Cancer

    Journal: Cell reports

    doi: 10.1016/j.celrep.2017.10.074

    A) Clear cell renal cell carcinoma (ccRCC) tumors, (T) and adjacent normal tissues (N) were stained with haematoxylin and eosin (H E). Bar scale represents 200µm. B) Proteins were extracted from above tumors and adjacent normal tissues and expression of PP5, CK1δ, VHL 30 and Hsp90 was examined by immunoblotting. Hsp90 was used a loading control. C) PP5, Cdc37 and phosphorylated S13-Cdc37, GR and phosphorylated S211-GR, CK1δ and VHL 30 proteins from ccRCC cell lines 786-O, A-498 (VHL-deficient) and Caki-1 (VHL-containing) were assessed by immunoblotting. GAPDH was used as a loading control. D) PP5 was silenced by siRNA in 786-O and Caki-1 cells. siCtrl represents the non-targeting siRNA control. Induction of apoptotic markers shown by immunoblotting using anti-cleaved caspase-3 and cleaved-PARP antibodies. GAPDH was used a loading control. E) Targeted siRNA was used to silence PP5 in A498 cells. siCtrl represents the non-targeting siRNA control. Induction of apoptotic markers shown by immunoblotting using anti-cleaved caspase-3 and cleaved-PARP antibodies. PP5 protein levels were also examined by immunoblotting. GAPDH was used as a control. F) PP5 was immunoprecipitated (IP) from ccRCC cell lines, Caki-1 and 786-O, lysates using anti-PP5 antibody or IgG (control). Threonine phosphorylation of PP5 was assessed by immunoblotting with anti-phosphothreonine antibody. GAPDH was used a loading control. G) PP5 was isolated from the lysates of 786-O cell treated with 2 µM IC261 for 16 hr using anti-PP5 or IgG (control) antibodies. Threonine phosphorylation of PP5 was examined by immunoblotting using anti-phosphothreonine antibody. Anti-phosphoserine antibody was used as a control.
    Figure Legend Snippet: A) Clear cell renal cell carcinoma (ccRCC) tumors, (T) and adjacent normal tissues (N) were stained with haematoxylin and eosin (H E). Bar scale represents 200µm. B) Proteins were extracted from above tumors and adjacent normal tissues and expression of PP5, CK1δ, VHL 30 and Hsp90 was examined by immunoblotting. Hsp90 was used a loading control. C) PP5, Cdc37 and phosphorylated S13-Cdc37, GR and phosphorylated S211-GR, CK1δ and VHL 30 proteins from ccRCC cell lines 786-O, A-498 (VHL-deficient) and Caki-1 (VHL-containing) were assessed by immunoblotting. GAPDH was used as a loading control. D) PP5 was silenced by siRNA in 786-O and Caki-1 cells. siCtrl represents the non-targeting siRNA control. Induction of apoptotic markers shown by immunoblotting using anti-cleaved caspase-3 and cleaved-PARP antibodies. GAPDH was used a loading control. E) Targeted siRNA was used to silence PP5 in A498 cells. siCtrl represents the non-targeting siRNA control. Induction of apoptotic markers shown by immunoblotting using anti-cleaved caspase-3 and cleaved-PARP antibodies. PP5 protein levels were also examined by immunoblotting. GAPDH was used as a control. F) PP5 was immunoprecipitated (IP) from ccRCC cell lines, Caki-1 and 786-O, lysates using anti-PP5 antibody or IgG (control). Threonine phosphorylation of PP5 was assessed by immunoblotting with anti-phosphothreonine antibody. GAPDH was used a loading control. G) PP5 was isolated from the lysates of 786-O cell treated with 2 µM IC261 for 16 hr using anti-PP5 or IgG (control) antibodies. Threonine phosphorylation of PP5 was examined by immunoblotting using anti-phosphothreonine antibody. Anti-phosphoserine antibody was used as a control.

    Techniques Used: Staining, Expressing, Immunoprecipitation, Isolation

    A) Dephosphorylation of phospho-S13-Cdc37 with a recombinant wild-type PP5-His 6 and phosphomimetic T362E-PP5-His 6 , in the presence of Hsp90α. Rate of Cdc37 dephosphorylation was assessed by immunoblotting with a phospho-specific S13-Cdc37 antibody over time (minutes). B) Recombinant wild-type PP5-His 6 was phosphorylated by CK1δ in vitro and then used in the dephosphorylation of phospho-S13-Cdc37 in vitro . The assay was performed in presence of Hsp90α. PP5 activity was assessed with immunoblotting using a phospho-specific S13-Cdc37 antibody over time (minutes). C) Dephosphorylation of phospho-S13-Cdc37 with recombinant wild-type PP5-His 6 and phosphomimetic T362E-PP5-His 6 was performed in the absence of Hsp90α. Activity was assessed with immunoblotting using a phospho-specific S13-Cdc37 antibody over time (minutes). D) Recombinant wild-type PP5-His 6 was phosphorylated by CK1δ in vitro and then used in the dephosphorylation of phospho-S13-Cdc37 in vitro without Hsp90α. PP5 activity was assessed with immunoblotting using a phospho-specific S13-Cdc37 antibody over time (minutes). E) Recombinant PP5-His 6 was used in an in vitro kinase assay with CK1δ-GST. PP5-His 6 was immunoprecipitated (IP) and threonine phosphorylation of PP5 as well as co-immunoprecipitation (Co-IP) of CK1δ-GST were examined by immunoblotting with anti-phosphothreonine and anti-GST antibodies. F) Recombinant wild-type PP5-His 6 was phosphorylated by CK1δ in vitro in the presence (+) or absence (−) of ATP. PP5-His 6 proteins were then used in the dephosphorylation of phospho-S13-Cdc37 in vitro without Hsp90α. PP5 activity was assessed with immunoblotting using a phospho-specific S13-Cdc37 antibody over time (minutes). G) PP5-FLAG and T362-PP5 phosphomutants (T362A and T362E) were transiently transfected in HEK293 cells. Cdc37, phospho-S13-Cdc37, GR and phospho S211-GR protein levels were examined by immunoblotting. Empty vector (EV) was used as a control, GAPDH was used a loading control. H) Wild-type PP5-FLAG, non-phosphorylating T362A-PP5-FLAG and the phosphomimetic T362E-PP5-FLAG were transiently expressed and IP from HEK293 cells. Co-IP of GR, Cdc37 and Hsp90 were examined by immunoblotting.
    Figure Legend Snippet: A) Dephosphorylation of phospho-S13-Cdc37 with a recombinant wild-type PP5-His 6 and phosphomimetic T362E-PP5-His 6 , in the presence of Hsp90α. Rate of Cdc37 dephosphorylation was assessed by immunoblotting with a phospho-specific S13-Cdc37 antibody over time (minutes). B) Recombinant wild-type PP5-His 6 was phosphorylated by CK1δ in vitro and then used in the dephosphorylation of phospho-S13-Cdc37 in vitro . The assay was performed in presence of Hsp90α. PP5 activity was assessed with immunoblotting using a phospho-specific S13-Cdc37 antibody over time (minutes). C) Dephosphorylation of phospho-S13-Cdc37 with recombinant wild-type PP5-His 6 and phosphomimetic T362E-PP5-His 6 was performed in the absence of Hsp90α. Activity was assessed with immunoblotting using a phospho-specific S13-Cdc37 antibody over time (minutes). D) Recombinant wild-type PP5-His 6 was phosphorylated by CK1δ in vitro and then used in the dephosphorylation of phospho-S13-Cdc37 in vitro without Hsp90α. PP5 activity was assessed with immunoblotting using a phospho-specific S13-Cdc37 antibody over time (minutes). E) Recombinant PP5-His 6 was used in an in vitro kinase assay with CK1δ-GST. PP5-His 6 was immunoprecipitated (IP) and threonine phosphorylation of PP5 as well as co-immunoprecipitation (Co-IP) of CK1δ-GST were examined by immunoblotting with anti-phosphothreonine and anti-GST antibodies. F) Recombinant wild-type PP5-His 6 was phosphorylated by CK1δ in vitro in the presence (+) or absence (−) of ATP. PP5-His 6 proteins were then used in the dephosphorylation of phospho-S13-Cdc37 in vitro without Hsp90α. PP5 activity was assessed with immunoblotting using a phospho-specific S13-Cdc37 antibody over time (minutes). G) PP5-FLAG and T362-PP5 phosphomutants (T362A and T362E) were transiently transfected in HEK293 cells. Cdc37, phospho-S13-Cdc37, GR and phospho S211-GR protein levels were examined by immunoblotting. Empty vector (EV) was used as a control, GAPDH was used a loading control. H) Wild-type PP5-FLAG, non-phosphorylating T362A-PP5-FLAG and the phosphomimetic T362E-PP5-FLAG were transiently expressed and IP from HEK293 cells. Co-IP of GR, Cdc37 and Hsp90 were examined by immunoblotting.

    Techniques Used: De-Phosphorylation Assay, Recombinant, In Vitro, Activity Assay, Kinase Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation

    A) Endogenous PP5 was immunoprecipitated (IP) from HEK293 cells and co-immunoprecipitation (Co-IP) of VHL 30 was assessed by immunoblotting. B) Endogenous VHL 30 was IP from HEK293 cells and Co-IP of PP5 was examined by immunoblotting. C) VHL 30 -FLAG or empty vector (EV) was transiently over-expressed in 786-O cells and endogenous PP5 protein levels were assessed by immunoblotting. GAPDH was used a loading control. D) The 786-O cells transiently expressing PP5-FLAG, were treated with or without 50 nM proteasome inhibitor bortezomib (BZ) for 2 hr. PP5-FLAG was also co-expressed with VHL 30 -His 6 with additional treatment of 50 nM BZ for 2 hr. PP5-FLAG was IP and its ubiquitination was assessed by immunoblotting. E) Egln1-HA, Egln2-HA, Egln3-HA and empty vector pcDNA3.1 were transiently over-expressed in HEK293 cells. The expression of Egln1, 2 and 3 as well as PP5, and HIF1α were assessed by immunoblotting with anti-HA, anti-PP5 and anti- HIF1α antibodies. GAPDH was used a loading control. F) Caki-1 cells cultured in normoxia and hypoxia (1%O 2 , 5%CO 2 , 94%N 2 ). PP5 and HIF1α protein levels were examined by immunoblotting using anti-PP5 and anti-HIF1α antibodies. GAPDH was used a loading control. G) HIF1α or HIF2α were silenced by small interfering RNA (siRNA) in HEK293 cells. HIF1α, HIF2α and PP5 protein levels were examined by immunoblotting using anti-HIF1α, anti-HIF2α and anti-PP5 antibodies. GAPDH was used a loading control.
    Figure Legend Snippet: A) Endogenous PP5 was immunoprecipitated (IP) from HEK293 cells and co-immunoprecipitation (Co-IP) of VHL 30 was assessed by immunoblotting. B) Endogenous VHL 30 was IP from HEK293 cells and Co-IP of PP5 was examined by immunoblotting. C) VHL 30 -FLAG or empty vector (EV) was transiently over-expressed in 786-O cells and endogenous PP5 protein levels were assessed by immunoblotting. GAPDH was used a loading control. D) The 786-O cells transiently expressing PP5-FLAG, were treated with or without 50 nM proteasome inhibitor bortezomib (BZ) for 2 hr. PP5-FLAG was also co-expressed with VHL 30 -His 6 with additional treatment of 50 nM BZ for 2 hr. PP5-FLAG was IP and its ubiquitination was assessed by immunoblotting. E) Egln1-HA, Egln2-HA, Egln3-HA and empty vector pcDNA3.1 were transiently over-expressed in HEK293 cells. The expression of Egln1, 2 and 3 as well as PP5, and HIF1α were assessed by immunoblotting with anti-HA, anti-PP5 and anti- HIF1α antibodies. GAPDH was used a loading control. F) Caki-1 cells cultured in normoxia and hypoxia (1%O 2 , 5%CO 2 , 94%N 2 ). PP5 and HIF1α protein levels were examined by immunoblotting using anti-PP5 and anti-HIF1α antibodies. GAPDH was used a loading control. G) HIF1α or HIF2α were silenced by small interfering RNA (siRNA) in HEK293 cells. HIF1α, HIF2α and PP5 protein levels were examined by immunoblotting using anti-HIF1α, anti-HIF2α and anti-PP5 antibodies. GAPDH was used a loading control.

    Techniques Used: Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing, Cell Culture, Small Interfering RNA

    A) ccRCC cell lines A498, 786-O and Caki-1 were treated with indicated amounts of CK1δ inhibitor IC261 for 16 hr and induction of apoptotic markers shown by immunoblotting using anti-cleaved caspase-3 and cleaved-PARP antibodies. GAPDH was used a loading control. B) AV/PI graphs of Caki-1, A498 and 786-O cells untreated (0µM) or treated with 2µM IC261 for 16hr for 2 hr. The top left quadrants represent dead cells stained only with PI. The bottom right quadrants represent apoptotic cells stained only with AV. The top right quadrants represent cells stained with both PI and AV (secondary necrosis and late apoptosis). Percentage of each stained cell population is indicated. Dot plots shown are representative of one of three independent experiments. C) MTT assay of A498, 786-O and Caki-1 cells treated with indicated amounts of IC261. The errors bars represent the SD of three independent experiments (****p
    Figure Legend Snippet: A) ccRCC cell lines A498, 786-O and Caki-1 were treated with indicated amounts of CK1δ inhibitor IC261 for 16 hr and induction of apoptotic markers shown by immunoblotting using anti-cleaved caspase-3 and cleaved-PARP antibodies. GAPDH was used a loading control. B) AV/PI graphs of Caki-1, A498 and 786-O cells untreated (0µM) or treated with 2µM IC261 for 16hr for 2 hr. The top left quadrants represent dead cells stained only with PI. The bottom right quadrants represent apoptotic cells stained only with AV. The top right quadrants represent cells stained with both PI and AV (secondary necrosis and late apoptosis). Percentage of each stained cell population is indicated. Dot plots shown are representative of one of three independent experiments. C) MTT assay of A498, 786-O and Caki-1 cells treated with indicated amounts of IC261. The errors bars represent the SD of three independent experiments (****p

    Techniques Used: Staining, MTT Assay

    37) Product Images from "Phosphorylation of EB1 regulates the recruitment of CLIP-170 and p150glued to the plus ends of astral microtubules"

    Article Title: Phosphorylation of EB1 regulates the recruitment of CLIP-170 and p150glued to the plus ends of astral microtubules

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14222

    Phosphorylation of EB1 by ASK1 promotes its interaction with CLIP-170 and p150 glued A . Cells were transfected with expression vectors carrying HA-EB1 WT, 3A, or 3D and then washed in a buffer that prevents microtubule deploymerization. Polymeric and soluble tubulin fractions were then prepared. Levels of CLIP-170 and p150 glued present in the polymeric and soluble fractions were examined by immunoblotting. B . Cells were transfected with expression vectors carrying HA-EB1 WT, 3A, or 3D. HA-EB1 proteins were immunoprecipitated (IP) from cell lysates, and immunoprecipitates were analyzed for the presence of p150 glued and CLIP-170 by immunoblotting with the indicated antibodies. C . In vitro kinase assays were performed using ASK1 or ASK1 kinase dead mutant (KD) immunoprecipitated from cells, together with bacterially purified His-EB1. Phosphorylated His-EB1 was affinity isolated and then incubated with in vitro translated p150 glued or CLIP-170. Ni-NTA pulldown (PD) was then performed, and the precipitates were immunoblotted with CLIP-170 or p150 glued antibodies.
    Figure Legend Snippet: Phosphorylation of EB1 by ASK1 promotes its interaction with CLIP-170 and p150 glued A . Cells were transfected with expression vectors carrying HA-EB1 WT, 3A, or 3D and then washed in a buffer that prevents microtubule deploymerization. Polymeric and soluble tubulin fractions were then prepared. Levels of CLIP-170 and p150 glued present in the polymeric and soluble fractions were examined by immunoblotting. B . Cells were transfected with expression vectors carrying HA-EB1 WT, 3A, or 3D. HA-EB1 proteins were immunoprecipitated (IP) from cell lysates, and immunoprecipitates were analyzed for the presence of p150 glued and CLIP-170 by immunoblotting with the indicated antibodies. C . In vitro kinase assays were performed using ASK1 or ASK1 kinase dead mutant (KD) immunoprecipitated from cells, together with bacterially purified His-EB1. Phosphorylated His-EB1 was affinity isolated and then incubated with in vitro translated p150 glued or CLIP-170. Ni-NTA pulldown (PD) was then performed, and the precipitates were immunoblotted with CLIP-170 or p150 glued antibodies.

    Techniques Used: Cross-linking Immunoprecipitation, Transfection, Expressing, Immunoprecipitation, In Vitro, Mutagenesis, Purification, Isolation, Incubation

    38) Product Images from "PHGDH as a key enzyme for serine biosynthesis in HIF2α-targeting therapy for renal cell carcinoma"

    Article Title: PHGDH as a key enzyme for serine biosynthesis in HIF2α-targeting therapy for renal cell carcinoma

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-17-1589

    PHGDH over expression in 786-o and A498 parental cells A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in 786-o and A498 cells. B, Representative images of parental and PHGDH overexpressed 786-o or A498 cells. C, Immunofluorescence analysis; PHGDH overexpressed 786-o or A498 cells showed morphological changes from a spindle to a round cell shape. The graph showed the ratio between spindle and round cells in PHGDH overexpressed 786-o or A498 cells. D, Representative image of colony formation in parental and PHGDH overexpressed 786-o or A498 cells. The graph showed the ratio of number of colonies between parental and PHGDH overexpressed cells (* P
    Figure Legend Snippet: PHGDH over expression in 786-o and A498 parental cells A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in 786-o and A498 cells. B, Representative images of parental and PHGDH overexpressed 786-o or A498 cells. C, Immunofluorescence analysis; PHGDH overexpressed 786-o or A498 cells showed morphological changes from a spindle to a round cell shape. The graph showed the ratio between spindle and round cells in PHGDH overexpressed 786-o or A498 cells. D, Representative image of colony formation in parental and PHGDH overexpressed 786-o or A498 cells. The graph showed the ratio of number of colonies between parental and PHGDH overexpressed cells (* P

    Techniques Used: Over Expression, Expressing, Immunofluorescence

    Establishment of HIF2α knock out SU-R-786-o cells A, Immunoblotting analysis showed that HIF2α was significantly depleted in sunitinib resistant 786-o cells. B, RSEM values of HIF targets genes from RNA-seq expression data between 786-o empty and HIF2α-KO-SU-R-786-o cells. C, Representative images of HIF2α-KO-SU-R-786-o cells. D, Cell proliferation assay between control and HIF2α-KO-SU-R-786-o (* P
    Figure Legend Snippet: Establishment of HIF2α knock out SU-R-786-o cells A, Immunoblotting analysis showed that HIF2α was significantly depleted in sunitinib resistant 786-o cells. B, RSEM values of HIF targets genes from RNA-seq expression data between 786-o empty and HIF2α-KO-SU-R-786-o cells. C, Representative images of HIF2α-KO-SU-R-786-o cells. D, Cell proliferation assay between control and HIF2α-KO-SU-R-786-o (* P

    Techniques Used: Knock-Out, RNA Sequencing Assay, Expressing, Proliferation Assay

    PHGDH inhibition by si-RNA and inhibitor A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in HIF2α-KO-SU-R-786-o cells. B, Cell proliferation assay by PHGDH si-RNA. C, Cell proliferation assay by PHGDH inhibitor (CBR-5884). (* P
    Figure Legend Snippet: PHGDH inhibition by si-RNA and inhibitor A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in HIF2α-KO-SU-R-786-o cells. B, Cell proliferation assay by PHGDH si-RNA. C, Cell proliferation assay by PHGDH inhibitor (CBR-5884). (* P

    Techniques Used: Inhibition, Expressing, Proliferation Assay

    39) Product Images from "A proteomic study of mitotic phase-specific interactors of EB1 reveals a role for SXIP-mediated protein interactions in anaphase onset"

    Article Title: A proteomic study of mitotic phase-specific interactors of EB1 reveals a role for SXIP-mediated protein interactions in anaphase onset

    Journal: Biology Open

    doi: 10.1242/bio.201410413

    Co-depletion of EB1 and EB3 delays anaphase onset more severely than chromosome congression. (A) Schematic describing siRNA and aphidicolin treatments in HeLa H2B-GFP mCherry-Tubulin cells. Cells were transfected with siRNA twice, at 72 h and 48 h prior to imaging. For synchronization, cells were treated with aphidicolin for 24 h, and then released into drug free medium for 8 h prior to imaging. At the end of imaging session, cell lysates were collected for immunoblotting to assess protein depletion extent. (B) Representative images from time-lapse movies of HeLa H2B-GFP; mCherry-Tubulin cells treated with both EB1 and EB3 or Control siRNA oligos as in (A). Scale bar: 10 µm. (C) Representative immunoblots showing the co-depletion of EB1 and EB3 in lysates collected from time-lapse imaging studies as shown in (B). Lysates of HeLa H2B-GFP; mCherry-Tubulin cells treated with indicated siRNA were processed for immunoblotting with α-γTubulin, α-EB1 and α-EB3 antibodies. (D-E) Cumulative frequency distribution graphs showing the time of chromosome congression (D) and anaphase onset (E) from NEBD in HeLa H2B-GFP; mCherry-Tubulin cells treated with control alone or EB1 and EB3 siRNA oligos. The graphs present the average values of two independent experiments with the error bars showing SEM values. n refers to number of cells. (F) Cartoon illustrating the loss of EB binding with its partners in the presence of excess SKAP (i), but not SKAP (NN) mutant (ii) that is defective for EB interaction. Excess of SKAP-EB interaction results in mitotic arrest, and this model highlights the importance of regulated plus-end complex interactions for controlling the timing of anaphase onset.
    Figure Legend Snippet: Co-depletion of EB1 and EB3 delays anaphase onset more severely than chromosome congression. (A) Schematic describing siRNA and aphidicolin treatments in HeLa H2B-GFP mCherry-Tubulin cells. Cells were transfected with siRNA twice, at 72 h and 48 h prior to imaging. For synchronization, cells were treated with aphidicolin for 24 h, and then released into drug free medium for 8 h prior to imaging. At the end of imaging session, cell lysates were collected for immunoblotting to assess protein depletion extent. (B) Representative images from time-lapse movies of HeLa H2B-GFP; mCherry-Tubulin cells treated with both EB1 and EB3 or Control siRNA oligos as in (A). Scale bar: 10 µm. (C) Representative immunoblots showing the co-depletion of EB1 and EB3 in lysates collected from time-lapse imaging studies as shown in (B). Lysates of HeLa H2B-GFP; mCherry-Tubulin cells treated with indicated siRNA were processed for immunoblotting with α-γTubulin, α-EB1 and α-EB3 antibodies. (D-E) Cumulative frequency distribution graphs showing the time of chromosome congression (D) and anaphase onset (E) from NEBD in HeLa H2B-GFP; mCherry-Tubulin cells treated with control alone or EB1 and EB3 siRNA oligos. The graphs present the average values of two independent experiments with the error bars showing SEM values. n refers to number of cells. (F) Cartoon illustrating the loss of EB binding with its partners in the presence of excess SKAP (i), but not SKAP (NN) mutant (ii) that is defective for EB interaction. Excess of SKAP-EB interaction results in mitotic arrest, and this model highlights the importance of regulated plus-end complex interactions for controlling the timing of anaphase onset.

    Techniques Used: Transfection, Imaging, Western Blot, Binding Assay, Mutagenesis

    40) Product Images from "Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC"

    Article Title: Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00535-16

    Disulfide bonding between OpmH His -Cys and TriA-Cys or TriB-Cys variants. (A) Immunoblotting analyses of membrane fractions isolated from P. aeruginosa cells producing the combinations of OpmH His -Cys and TriA-Cys or TriB-Cys variants indicated. OpmH His
    Figure Legend Snippet: Disulfide bonding between OpmH His -Cys and TriA-Cys or TriB-Cys variants. (A) Immunoblotting analyses of membrane fractions isolated from P. aeruginosa cells producing the combinations of OpmH His -Cys and TriA-Cys or TriB-Cys variants indicated. OpmH His

    Techniques Used: Isolation

    Related Articles

    Transfection:

    Article Title: Repetitive N-WASP-Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly
    Article Snippet: .. Immunoblotting To prepare mammalian cell lysates, transfected cells were collected in PBS+2 mM EDTA and lysed in 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% Triton X-100, 1 mM Na3 VO4 , 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, pepstatin, and chymostatin (Sigma)), prior to mixing with SDS-PAGE sample buffer. .. Protein samples were boiled for 10 minutes, centrifuged, and analyzed by 10% SDS-PAGE prior to staining with Coomassie blue or transferring to nitrocellulose membranes and staining with Ponceau S. Membranes were blocked in PBS+5% milk (PBSM) before probing with N-WASP or GFP antibodies, as described previously .

    Protease Inhibitor:

    Article Title: MP470, a novel receptor tyrosine kinase inhibitor, in combination with Erlotinib inhibits the HER family/PI3K/Akt pathway and tumor growth in prostate cancer
    Article Snippet: .. Immunoblotting The cells were lysed in NP-40 lysis buffer containing 50 mM Tris.Cl (pH 7.4), 0.15 M NaCl, 0.5% NP-40, 1 mM DTT, 50 mM Sodium Fluoride, and 2 μl/ml Protease inhibitor cocktail (Sigma, St. Louis, MO). .. Protein concentrations were determined using the BioRad protein assay kit (Hercules, CA) and 50 μg of protein was resolved by electrophoresis on a 10% SDS-PAGE gel.

    Article Title: MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases
    Article Snippet: .. ImmunoblottingThe pellets containing nanoparticles were lysed in RIPA-buffer containing Protease Inhibitor Cocktail (Sigma-Aldrich) and Phosphatase Inhibitor Cocktail (nakarai tesque, Kyoto, Japan). .. The concentrations of protein were quantified by performing the Bradford protein assay (DC Protein assay kit, Bio-Rad, Hercules, CA, USA).

    Acrylamide Gel Assay:

    Article Title: EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity
    Article Snippet: .. For immunoblotting, proteins were separated by SDS-PAGE (10% acrylamide gel) and transferred to PVDF membrane (Millipore) by electro-transfer at 80 V for 90 min. .. The membrane was blocked in 1× TBS buffer containing 5% skim milk powder and further incubated with primary antibody and secondary antibody.

    Incubation:

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *
    Article Snippet: .. TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling). .. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.

    Expressing:

    Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells
    Article Snippet: .. Immunoblotting To analyze protein expression, BeWo cells and primary vCTB were rinsed with PBS and lysed with ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris-HCl pH 7.4, 1% NP-40, 0,25% Na-deoxycholate, 150 mmol/l NaCl and 1 mmol/l EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich). .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Waltham, MA).

    Modification:

    Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells
    Article Snippet: .. Immunoblotting To analyze protein expression, BeWo cells and primary vCTB were rinsed with PBS and lysed with ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris-HCl pH 7.4, 1% NP-40, 0,25% Na-deoxycholate, 150 mmol/l NaCl and 1 mmol/l EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich). .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Waltham, MA).

    Lysis:

    Article Title: MP470, a novel receptor tyrosine kinase inhibitor, in combination with Erlotinib inhibits the HER family/PI3K/Akt pathway and tumor growth in prostate cancer
    Article Snippet: .. Immunoblotting The cells were lysed in NP-40 lysis buffer containing 50 mM Tris.Cl (pH 7.4), 0.15 M NaCl, 0.5% NP-40, 1 mM DTT, 50 mM Sodium Fluoride, and 2 μl/ml Protease inhibitor cocktail (Sigma, St. Louis, MO). .. Protein concentrations were determined using the BioRad protein assay kit (Hercules, CA) and 50 μg of protein was resolved by electrophoresis on a 10% SDS-PAGE gel.

    SDS Page:

    Article Title: Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus
    Article Snippet: .. For immunoblotting, proteins were separated by SDS-PAGE and transferred to 0.45 μm PVDF membranes (Millipore). .. The membranes were blocked with 5 % (w/v) skimmed-milk prepared in Tris-buffered saline-0.05 % Tween 20 (TBS-T) for 1 h at room temperature, followed by incubation with primary antibody diluted in 3 % (w/v) skimmed-milk in TBS-T at 4 °C overnight.

    Article Title: EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity
    Article Snippet: .. For immunoblotting, proteins were separated by SDS-PAGE (10% acrylamide gel) and transferred to PVDF membrane (Millipore) by electro-transfer at 80 V for 90 min. .. The membrane was blocked in 1× TBS buffer containing 5% skim milk powder and further incubated with primary antibody and secondary antibody.

    Article Title: Inflammation and Hyperglycemia Mediate Deaf1 Splicing in the Pancreatic Lymph Nodes via Distinct Pathways During Type 1 Diabetes
    Article Snippet: .. SDS-PAGE and immunoblotting were performed according to standard procedures using the rabbit polyclonal anti-PTBP2 (ABE431; Millipore) at 1:2,000 and polyclonal anti-FUSIP1/SRSF10 (E-23; sc-101961; Santa Cruz Biotechnology) at 1:1,000. ..

    Article Title: Repetitive N-WASP-Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly
    Article Snippet: .. Immunoblotting To prepare mammalian cell lysates, transfected cells were collected in PBS+2 mM EDTA and lysed in 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% Triton X-100, 1 mM Na3 VO4 , 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, pepstatin, and chymostatin (Sigma)), prior to mixing with SDS-PAGE sample buffer. .. Protein samples were boiled for 10 minutes, centrifuged, and analyzed by 10% SDS-PAGE prior to staining with Coomassie blue or transferring to nitrocellulose membranes and staining with Ponceau S. Membranes were blocked in PBS+5% milk (PBSM) before probing with N-WASP or GFP antibodies, as described previously .

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  • 93
    Millipore immunoblotting analysis
    Specificity of antihuman l ‐type amino acid transporter 1 ( LAT 1) mA bs in human and macaca cells. A, ACHN human and MK .P3 macaca cells were stained with antihuman LAT 1 mA bs (Ab1 or Ab2), followed by phycoerythrin ( PE )‐conjugated antirat IgG, and analyzed by flow cytometry (FCM). Values indicate the ratio (+ mA b/− mA b) of mean fluorescence intensity ( rMFI ). B, Effects of RNA interference on the expression of human LAT 1 and CD 98 heavy chain ( CD 98hc) proteins in human and macaca cells were analyzed. Cells were treated with mock (scramble) or LAT 1 si RNA (#1, #2 or #3) for 72 h, stained with rat mA bs against human LAT 1 (Ab1) or CD 98hc ( HR 35), followed by PE ‐conjugated antirat IgG, and analyzed for protein expression by FCM . C, Antihuman LAT 1 mA b (Ab1) was evaluated for its reactivity against RH 7777 rat cells expressing macaca (left) or human (right) LAT 1‐ GFP . D, Association of introduced macaca LAT 1‐ GFP with endogenous rat or human CD 98hc was examined. Cell lysates from RH 7777 (upper) or HEK 293 (lower) expressing macaca LAT 1 were subjected to immunoprecipitation with control IgG, anti‐ LAT 1, or anti‐ CD 98hc mA b, and the resulting precipitates, as well as the original cell lysates (input), were subjected to <t>immunoblotting</t> with anti‐GFP rabbit polyclonal antibodies
    Immunoblotting Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore immunoblot analysis immunoblotting
    BMI1 suppresses cell death by stabilizing cyclin E1. a , <t>Immunoblot</t> analysis of cyclins and CDKs in pooled BMI1-sensitive clones following BMI1 knockdown. α-tubulin levels are shown as loading control. b , Immunoblot analysis of cyclin E1 levels in BE(2)-C cells either uninfected (parental) or infected with retroviruses expressing shRNA sequences against GFP (GFPsh) or different regions of CCNE1 (CCNE1sh-80 and -81). Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the cyclin E1 level in parental cells. c , Crystal violet staining of BE(2)-C cells expressing either GFPsh or CCNE1sh-80. d , Immunoblot analysis of BMI1 and cyclin E1 levels in pooled BMI1-sensitive clones infected with retroviruses expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 3 days. α-tubulin levels are shown as loading control. e , Phase contrast imaging of pooled BMI1-sensitive clones expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 6 days. Scale bars, 100 μm. f , qRT-PCR analysis of CCND1 and CCNE1 mRNA levels in pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days (error bars, s.d., n=3). g , Quantification of cyclin E1 half-life in pooled BMI1-sensitive clones cultured in the presence or absence of Doxy for 3 days. Samples were collected at various time points following addition of cycloheximide (CHX) for immunoblot analysis. Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the initial levels at time zero (error bars, s.d., n=4). h , In vivo ubiquitination assay of pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days and co-transfected with Flag-ubiquitin and Myc-cyclin E1 expression plasmids. Polyubiquitinated cyclin E1 was detected by immunoprecipitation of Myccyclin E1, followed by <t>immunoblotting</t> for Flag-ubiquitin.
    Immunoblot Analysis Immunoblotting, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore ebv gene expression immunoblotting
    Analysis of early events of in vitro B cell transformation. Primary B cells were isolated and infected with the lytic cycle-deficient BZKO rEBV. (A) Map of the latent BHRF1 transcript expressed in LCLs showing the positions of the primers (black arrows) and probe (black box) sequences used to detect W2-BHRF1 and Y2-BHRF1 spliced transcripts by QRT-PCR. (B) Analysis of gene expression using QRT-PCR assays to measure Wp and Cp activity and latent W2-BHRF1, Y2-BHRF1 spliced, and LMP1 transcripts at time points (0 to 120 hrs) following <t>EBV</t> infection. Results, expressed as in Figure 3B , are shown as the mean+/−SD of triplicate readings from a representative experiment. Similar patterns of results were observed in two further infection experiments. (C) <t>Immunoblotting</t> to detect expression of EBNA-LP, EBNA2, BHRF1, and LMP1 at time points following EBV infection. The established LCL X50-7 was used as a positive control for EBNA-LP, EBNA-2, and LMP1 expression. Akata-BL cells induced into lytic replication by surface IgG cross-linking (60% cells in lytic cycle) was used as a positive control for lytic BHRF1 expression. An EBV-negative Awia-BL clone was used as a negative control throughout. Size markers are shown in kD. This result was confirmed in two further infection experiments. (D) CD4+ T cell recognition of HLA-DR4-positive primary B cells 4 and 8 days after infection with either the BZKO virus (lytic cycle-deficient) or with the BHRF1KO virus. Data are shown for (left panel) a DR4-restricted T cell clone specific for an EBNA2 epitope GQT, and (right panel) a DR4-restricted T cell clone specific for a BHRF1 epitope PYY. Results are expressed as IFNγ release into supernatant as measured by ELISA; values (mean+/−SD of triplicate readings) are shown for infected B cell targets (black bars) and for the same target cells pre-pulsed with the relevant epitope peptide (grey bars). A similar pattern of results was obtained in three successive experiments; there was never any recognition of DR4-mismatched targets included as controls in the same assays (data not shown).
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    Specificity of antihuman l ‐type amino acid transporter 1 ( LAT 1) mA bs in human and macaca cells. A, ACHN human and MK .P3 macaca cells were stained with antihuman LAT 1 mA bs (Ab1 or Ab2), followed by phycoerythrin ( PE )‐conjugated antirat IgG, and analyzed by flow cytometry (FCM). Values indicate the ratio (+ mA b/− mA b) of mean fluorescence intensity ( rMFI ). B, Effects of RNA interference on the expression of human LAT 1 and CD 98 heavy chain ( CD 98hc) proteins in human and macaca cells were analyzed. Cells were treated with mock (scramble) or LAT 1 si RNA (#1, #2 or #3) for 72 h, stained with rat mA bs against human LAT 1 (Ab1) or CD 98hc ( HR 35), followed by PE ‐conjugated antirat IgG, and analyzed for protein expression by FCM . C, Antihuman LAT 1 mA b (Ab1) was evaluated for its reactivity against RH 7777 rat cells expressing macaca (left) or human (right) LAT 1‐ GFP . D, Association of introduced macaca LAT 1‐ GFP with endogenous rat or human CD 98hc was examined. Cell lysates from RH 7777 (upper) or HEK 293 (lower) expressing macaca LAT 1 were subjected to immunoprecipitation with control IgG, anti‐ LAT 1, or anti‐ CD 98hc mA b, and the resulting precipitates, as well as the original cell lysates (input), were subjected to immunoblotting with anti‐GFP rabbit polyclonal antibodies

    Journal: Cancer Science

    Article Title: Anti‐tumor effects of mAb against l‐type amino acid transporter 1 ( LAT1) bound to human and monkey LAT1 with dual avidity modes, et al. Anti‐tumor effects of mAb to l ‐type amino acid transporter 1 (LAT1) bound to human and monkey LAT1 with dual avidity modes

    doi: 10.1111/cas.13908

    Figure Lengend Snippet: Specificity of antihuman l ‐type amino acid transporter 1 ( LAT 1) mA bs in human and macaca cells. A, ACHN human and MK .P3 macaca cells were stained with antihuman LAT 1 mA bs (Ab1 or Ab2), followed by phycoerythrin ( PE )‐conjugated antirat IgG, and analyzed by flow cytometry (FCM). Values indicate the ratio (+ mA b/− mA b) of mean fluorescence intensity ( rMFI ). B, Effects of RNA interference on the expression of human LAT 1 and CD 98 heavy chain ( CD 98hc) proteins in human and macaca cells were analyzed. Cells were treated with mock (scramble) or LAT 1 si RNA (#1, #2 or #3) for 72 h, stained with rat mA bs against human LAT 1 (Ab1) or CD 98hc ( HR 35), followed by PE ‐conjugated antirat IgG, and analyzed for protein expression by FCM . C, Antihuman LAT 1 mA b (Ab1) was evaluated for its reactivity against RH 7777 rat cells expressing macaca (left) or human (right) LAT 1‐ GFP . D, Association of introduced macaca LAT 1‐ GFP with endogenous rat or human CD 98hc was examined. Cell lysates from RH 7777 (upper) or HEK 293 (lower) expressing macaca LAT 1 were subjected to immunoprecipitation with control IgG, anti‐ LAT 1, or anti‐ CD 98hc mA b, and the resulting precipitates, as well as the original cell lysates (input), were subjected to immunoblotting with anti‐GFP rabbit polyclonal antibodies

    Article Snippet: For immunoblotting analysis, proteins were solubilized in loading buffer, subjected to SDS‐PAGE (8% gels) and transferred to Immobilon‐P (Millipore), and exposed to rabbit anti‐GFP pAb.

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence, Expressing, Immunoprecipitation

    Splicing factors are part of high molecular weight complexes. Crude cell extracts of R4T, RT2T, R6T, and R2T were subjected to size exclusion chromatography, and 18 fractions were analyzed by immunoblotting with antiserum against the TAP sequence. As a control, the large subunit of Rubisco, which has an estimated size of 560 kDa, was detected using an α-rbcL antibody. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.

    Journal: The Journal of Biological Chemistry

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *

    doi: 10.1074/jbc.M116.750570

    Figure Lengend Snippet: Splicing factors are part of high molecular weight complexes. Crude cell extracts of R4T, RT2T, R6T, and R2T were subjected to size exclusion chromatography, and 18 fractions were analyzed by immunoblotting with antiserum against the TAP sequence. As a control, the large subunit of Rubisco, which has an estimated size of 560 kDa, was detected using an α-rbcL antibody. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.

    Article Snippet: TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling).

    Techniques: Molecular Weight, Size-exclusion Chromatography, Sequencing, Filtration

    BMI1 suppresses cell death by stabilizing cyclin E1. a , Immunoblot analysis of cyclins and CDKs in pooled BMI1-sensitive clones following BMI1 knockdown. α-tubulin levels are shown as loading control. b , Immunoblot analysis of cyclin E1 levels in BE(2)-C cells either uninfected (parental) or infected with retroviruses expressing shRNA sequences against GFP (GFPsh) or different regions of CCNE1 (CCNE1sh-80 and -81). Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the cyclin E1 level in parental cells. c , Crystal violet staining of BE(2)-C cells expressing either GFPsh or CCNE1sh-80. d , Immunoblot analysis of BMI1 and cyclin E1 levels in pooled BMI1-sensitive clones infected with retroviruses expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 3 days. α-tubulin levels are shown as loading control. e , Phase contrast imaging of pooled BMI1-sensitive clones expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 6 days. Scale bars, 100 μm. f , qRT-PCR analysis of CCND1 and CCNE1 mRNA levels in pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days (error bars, s.d., n=3). g , Quantification of cyclin E1 half-life in pooled BMI1-sensitive clones cultured in the presence or absence of Doxy for 3 days. Samples were collected at various time points following addition of cycloheximide (CHX) for immunoblot analysis. Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the initial levels at time zero (error bars, s.d., n=4). h , In vivo ubiquitination assay of pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days and co-transfected with Flag-ubiquitin and Myc-cyclin E1 expression plasmids. Polyubiquitinated cyclin E1 was detected by immunoprecipitation of Myccyclin E1, followed by immunoblotting for Flag-ubiquitin.

    Journal: Oncogene

    Article Title: Cyclin E1 is a common target of BMI1 and MYCN and a prognostic marker for neuroblastoma progression

    doi: 10.1038/onc.2011.536

    Figure Lengend Snippet: BMI1 suppresses cell death by stabilizing cyclin E1. a , Immunoblot analysis of cyclins and CDKs in pooled BMI1-sensitive clones following BMI1 knockdown. α-tubulin levels are shown as loading control. b , Immunoblot analysis of cyclin E1 levels in BE(2)-C cells either uninfected (parental) or infected with retroviruses expressing shRNA sequences against GFP (GFPsh) or different regions of CCNE1 (CCNE1sh-80 and -81). Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the cyclin E1 level in parental cells. c , Crystal violet staining of BE(2)-C cells expressing either GFPsh or CCNE1sh-80. d , Immunoblot analysis of BMI1 and cyclin E1 levels in pooled BMI1-sensitive clones infected with retroviruses expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 3 days. α-tubulin levels are shown as loading control. e , Phase contrast imaging of pooled BMI1-sensitive clones expressing either GFP or Myc-cyclin E1 and cultured in the presence or absence of Doxy for 6 days. Scale bars, 100 μm. f , qRT-PCR analysis of CCND1 and CCNE1 mRNA levels in pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days (error bars, s.d., n=3). g , Quantification of cyclin E1 half-life in pooled BMI1-sensitive clones cultured in the presence or absence of Doxy for 3 days. Samples were collected at various time points following addition of cycloheximide (CHX) for immunoblot analysis. Cyclin E1 levels were quantified against α-tubulin and are presented as the fraction of the initial levels at time zero (error bars, s.d., n=4). h , In vivo ubiquitination assay of pooled BMI1-sensitive cells cultured in the presence or absence of Doxy for 3 days and co-transfected with Flag-ubiquitin and Myc-cyclin E1 expression plasmids. Polyubiquitinated cyclin E1 was detected by immunoprecipitation of Myccyclin E1, followed by immunoblotting for Flag-ubiquitin.

    Article Snippet: Immunoblot analysis Immunoblotting was conducted according to standard procedures using the following primary antibodies: mouse anti-BMI1 (05-637, 1:500; Upstate, Millipore, Billerica, MA), rabbit anti-cyclin A2 (sc-751, 1:200), mouse anti-cyclin B1 (sc-245, 1:200), mouse anti-cyclin D1 (sc-20044, 1:200), mouse anti-cyclin E1 (sc-56310, 1:200), rabbit anti-cyclin E2 (ab40890, 1:2000; Abcam, Cambridge, MA), mouse anti-Flag tag (F-4042, 1:2000; Sigma-Aldrich), mouse anti-MYCN (OP13, 1:200; Calbiochem, EMD Biosciences, San Diego, CA), mouse anti-Myc tag (9E10, hybridoma supernatant, 1:10), rabbit anti-p14ARF (A300-340A, 1:500; Bethyl Laboratories, Montgomery, TX), rabbit anti-p16Ink4a (sc-468, 1:200), rabbit anti-β-actin (600-401-886, 1:2000; Rockland Immunochemicals, Gilbertsville, PA), and mouse anti-α-tubulin (B-5-1-2, 1:5000; Sigma-Aldrich).

    Techniques: Clone Assay, Infection, Expressing, shRNA, Staining, Cell Culture, Imaging, Quantitative RT-PCR, In Vivo, Ubiquitin Assay, Transfection, Immunoprecipitation

    Analysis of early events of in vitro B cell transformation. Primary B cells were isolated and infected with the lytic cycle-deficient BZKO rEBV. (A) Map of the latent BHRF1 transcript expressed in LCLs showing the positions of the primers (black arrows) and probe (black box) sequences used to detect W2-BHRF1 and Y2-BHRF1 spliced transcripts by QRT-PCR. (B) Analysis of gene expression using QRT-PCR assays to measure Wp and Cp activity and latent W2-BHRF1, Y2-BHRF1 spliced, and LMP1 transcripts at time points (0 to 120 hrs) following EBV infection. Results, expressed as in Figure 3B , are shown as the mean+/−SD of triplicate readings from a representative experiment. Similar patterns of results were observed in two further infection experiments. (C) Immunoblotting to detect expression of EBNA-LP, EBNA2, BHRF1, and LMP1 at time points following EBV infection. The established LCL X50-7 was used as a positive control for EBNA-LP, EBNA-2, and LMP1 expression. Akata-BL cells induced into lytic replication by surface IgG cross-linking (60% cells in lytic cycle) was used as a positive control for lytic BHRF1 expression. An EBV-negative Awia-BL clone was used as a negative control throughout. Size markers are shown in kD. This result was confirmed in two further infection experiments. (D) CD4+ T cell recognition of HLA-DR4-positive primary B cells 4 and 8 days after infection with either the BZKO virus (lytic cycle-deficient) or with the BHRF1KO virus. Data are shown for (left panel) a DR4-restricted T cell clone specific for an EBNA2 epitope GQT, and (right panel) a DR4-restricted T cell clone specific for a BHRF1 epitope PYY. Results are expressed as IFNγ release into supernatant as measured by ELISA; values (mean+/−SD of triplicate readings) are shown for infected B cell targets (black bars) and for the same target cells pre-pulsed with the relevant epitope peptide (grey bars). A similar pattern of results was obtained in three successive experiments; there was never any recognition of DR4-mismatched targets included as controls in the same assays (data not shown).

    Journal: PLoS Pathogens

    Article Title: An Epstein-Barr Virus Anti-Apoptotic Protein Constitutively Expressed in Transformed Cells and Implicated in Burkitt Lymphomagenesis: The Wp/BHRF1 Link

    doi: 10.1371/journal.ppat.1000341

    Figure Lengend Snippet: Analysis of early events of in vitro B cell transformation. Primary B cells were isolated and infected with the lytic cycle-deficient BZKO rEBV. (A) Map of the latent BHRF1 transcript expressed in LCLs showing the positions of the primers (black arrows) and probe (black box) sequences used to detect W2-BHRF1 and Y2-BHRF1 spliced transcripts by QRT-PCR. (B) Analysis of gene expression using QRT-PCR assays to measure Wp and Cp activity and latent W2-BHRF1, Y2-BHRF1 spliced, and LMP1 transcripts at time points (0 to 120 hrs) following EBV infection. Results, expressed as in Figure 3B , are shown as the mean+/−SD of triplicate readings from a representative experiment. Similar patterns of results were observed in two further infection experiments. (C) Immunoblotting to detect expression of EBNA-LP, EBNA2, BHRF1, and LMP1 at time points following EBV infection. The established LCL X50-7 was used as a positive control for EBNA-LP, EBNA-2, and LMP1 expression. Akata-BL cells induced into lytic replication by surface IgG cross-linking (60% cells in lytic cycle) was used as a positive control for lytic BHRF1 expression. An EBV-negative Awia-BL clone was used as a negative control throughout. Size markers are shown in kD. This result was confirmed in two further infection experiments. (D) CD4+ T cell recognition of HLA-DR4-positive primary B cells 4 and 8 days after infection with either the BZKO virus (lytic cycle-deficient) or with the BHRF1KO virus. Data are shown for (left panel) a DR4-restricted T cell clone specific for an EBNA2 epitope GQT, and (right panel) a DR4-restricted T cell clone specific for a BHRF1 epitope PYY. Results are expressed as IFNγ release into supernatant as measured by ELISA; values (mean+/−SD of triplicate readings) are shown for infected B cell targets (black bars) and for the same target cells pre-pulsed with the relevant epitope peptide (grey bars). A similar pattern of results was obtained in three successive experiments; there was never any recognition of DR4-mismatched targets included as controls in the same assays (data not shown).

    Article Snippet: EBV gene expression Immunoblotting was carried out as described previously using mAbs to: EBNA1 (1H4), EBNA2 (PE2), EBNA3C (E3CA10), LMP1 (CS1-4), BZLF1 (BZ-1) (all used at dilutions of 1 in 50) ; BHRF1 (5B11: Millipore, used at a dilution of 1 in 1000), Calregulin (H-170, Santa-Cruz Biotechnology, used at a dilution of 1 in 1000) and polyclonal antibodies specific for EBNA3A and 3B (Exalpha Biologicals, Maynard, MA; the antibodies were used at a dilution of 1 in 1000 to detect EBNA3A and 1 in 500 to detect EBNA3B) and for PARP1 N-terminal region (H-300, Santa-Cruz Biotechnology, used at a dilution of 1 in 1000).

    Techniques: In Vitro, Transformation Assay, Isolation, Infection, Quantitative RT-PCR, Expressing, Activity Assay, Positive Control, Negative Control, Enzyme-linked Immunosorbent Assay