pvdf membrane  (Bio-Rad)

 
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    Name:
    Immun Blot PVDF Membrane
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    Pkg of 1 roll 0 2 µm 26 cm x 3 3 m bulk membrane for high binding 150 160 µg cm2 immunoblotting
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    1620177
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    Bio-Rad pvdf membrane
    Immun Blot PVDF Membrane
    Pkg of 1 roll 0 2 µm 26 cm x 3 3 m bulk membrane for high binding 150 160 µg cm2 immunoblotting
    https://www.bioz.com/result/pvdf membrane/product/Bio-Rad
    Average 99 stars, based on 3488 article reviews
    Price from $9.99 to $1999.99
    pvdf membrane - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Oxygen-responsive p53 tetramer-octamer switch controls cell fate"

    Article Title: Oxygen-responsive p53 tetramer-octamer switch controls cell fate

    Journal: bioRxiv

    doi: 10.1101/841668

    p53 T associates with HIF-1. (A) Anti-HIF-1α BN-PAGE immune-blot shows the rate of accumulation of different complexes of HIF-1α at 1% O 2 in HCT116p53+/+ and HCT116p53−/− cells. Purple arrows indicate HIF-1α species (M.W. 120kDa), yellow arrow shows HIF-1 complex (M.W. 212 kDa) and blue arrow suggests p53-HIF-1 complex (M.W. > HIF-1) after an extended run of lysates in 3-15% Bis-tris gradient gel. The black arrow shows higher-order HIF-1α species in HCT116p53+/+ cell line. (B) Foci like structures (yellow arrows) showing co-localization of exogenous HIF-1α (ECFP), HIF-1β (EYFP) and exogenous or endogenous p53 (DsRed Ex or TRITC) in the nucleus of the cell. Scale bar 100μm. (C) Sequestration of endogenous p53 by exogenous HIF-1 subunits in concentration-dependent manner. Scale bar 50μm. Fluorescence images are pseudo-colored and color calibration bars indicate pixel-wise fluorescence intensity. (D) Triple immune reaction-based identification of endogenous p53T-HIF-1 complex. Green arrows indicate complex with M.W. > p53-HIF-1. The black arrow identifies higher order HIF-1α species. Blue, magenta and yellow arrows indicate p53-HIF-1, p53T and HIF-1 complex respectively. Native protein standards were separated from the PVDF membrane post-transfer and stained separately by Coomassie G250. (E) Identification of endogenous p53-HIF-1 complex by cross-reaction of the same immune band against three antibodies by stepwise stripping. anti-p53 DO1 (cyan), anti-HIF-1α (green) and anti-HIF-1β (red) immune blots were merged cautiously in silico to detect cross-reactivity (white). (F) Effect of different detergent combinations on p53 or HIF-1α complexes. Blue arrows indicate p53-HIF-1 complex positions in the immune-blots. Anti-p53 immune-staining confirms dissociation of intact T from p53-HIF-1 complex by D2 detergent (magenta arrow). (G) Schematic representation of the principle of detergent displacement strategy (left panel). Anti-HIF-1α immune blot was stripped for anti-p53 immune detection and two immune blots were cautiously merged in silico to identify the dissociated p53T (magenta) and HIF-1(cyan) entities (dotted yellow circles) (right panel). Higher-order HIF-1α aggregates are shown by black arrows. For the merged anti-p53 immune-blot image, refer to Fig 6D . 3-15% Bis-Tris gradient gel was selected for proper resolution of all complexes in 1D and 2D BN-PAGE run.
    Figure Legend Snippet: p53 T associates with HIF-1. (A) Anti-HIF-1α BN-PAGE immune-blot shows the rate of accumulation of different complexes of HIF-1α at 1% O 2 in HCT116p53+/+ and HCT116p53−/− cells. Purple arrows indicate HIF-1α species (M.W. 120kDa), yellow arrow shows HIF-1 complex (M.W. 212 kDa) and blue arrow suggests p53-HIF-1 complex (M.W. > HIF-1) after an extended run of lysates in 3-15% Bis-tris gradient gel. The black arrow shows higher-order HIF-1α species in HCT116p53+/+ cell line. (B) Foci like structures (yellow arrows) showing co-localization of exogenous HIF-1α (ECFP), HIF-1β (EYFP) and exogenous or endogenous p53 (DsRed Ex or TRITC) in the nucleus of the cell. Scale bar 100μm. (C) Sequestration of endogenous p53 by exogenous HIF-1 subunits in concentration-dependent manner. Scale bar 50μm. Fluorescence images are pseudo-colored and color calibration bars indicate pixel-wise fluorescence intensity. (D) Triple immune reaction-based identification of endogenous p53T-HIF-1 complex. Green arrows indicate complex with M.W. > p53-HIF-1. The black arrow identifies higher order HIF-1α species. Blue, magenta and yellow arrows indicate p53-HIF-1, p53T and HIF-1 complex respectively. Native protein standards were separated from the PVDF membrane post-transfer and stained separately by Coomassie G250. (E) Identification of endogenous p53-HIF-1 complex by cross-reaction of the same immune band against three antibodies by stepwise stripping. anti-p53 DO1 (cyan), anti-HIF-1α (green) and anti-HIF-1β (red) immune blots were merged cautiously in silico to detect cross-reactivity (white). (F) Effect of different detergent combinations on p53 or HIF-1α complexes. Blue arrows indicate p53-HIF-1 complex positions in the immune-blots. Anti-p53 immune-staining confirms dissociation of intact T from p53-HIF-1 complex by D2 detergent (magenta arrow). (G) Schematic representation of the principle of detergent displacement strategy (left panel). Anti-HIF-1α immune blot was stripped for anti-p53 immune detection and two immune blots were cautiously merged in silico to identify the dissociated p53T (magenta) and HIF-1(cyan) entities (dotted yellow circles) (right panel). Higher-order HIF-1α aggregates are shown by black arrows. For the merged anti-p53 immune-blot image, refer to Fig 6D . 3-15% Bis-Tris gradient gel was selected for proper resolution of all complexes in 1D and 2D BN-PAGE run.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Concentration Assay, Fluorescence, Staining, Stripping Membranes, In Silico

    Metastable p53 T operates via an oxygen-sensitive T⇀O switch. (A) Schematic representation of the CHX trap in a hypoxia gradient. (B, C) To determine metastable p53 T dynamics in response to hypoxia, CHX trap design in (A) was used to capture p53 homo-oligomerization dynamics by anti-p53 BN-PAGE immune blotting at 1, 0.1 or 5% O 2 (immune blot is shown in Fig. 5B ). To sufficiently resolve each homo-oligomer (especially T and O) 5-15% Bis-tris gradient gel (pH 7.0) was utilized. T1 represents duration for which HCT116 p53+/+ cells were exposed to hypoxia before CHX treatment. Purple arrows indicate p53 pool segregated in its constituent homo-oligomers without CHX trap. T2 represents the duration of CHX for hypoxic cells. 24h > T2 > 6h was always maintained for p53T dynamics in 0-72h T1. A red arrow in (B) shows p53 aggregating smears. Native protein standards were run in the same gel and after transfer of samples on PVDF membrane; its lane was cut and stained separately with coomassie brilliant blue G250. Due to inclusion of protein standards in 15 well gel, 60 th h sample for 1% O 2 was analyzed separately or from other replicates. SDS-PAGE based analysis of total p53 pool and GAPDH loading control of immune blots in (B, C) is shown in Fig 5A, B or Fig S3F. (D) R.A. measurements from (B, C) show oxygen-sensitive p53T via shifts in equilibrium state (5% O 2 ). Green and magenta circles correspond to on-off pattern of p53 switch deciphered at 6h. The magenta arrow shows enhanced dimerization or octamerization via T during initial durations that initiates shifts at 1 and 0.1% O 2 respectively. Values and error bars in correspond to mean and standard deviation from three independent replicates of the experiment respectively and are best represented by the immune blots in (B, C) or Fig. 5B .
    Figure Legend Snippet: Metastable p53 T operates via an oxygen-sensitive T⇀O switch. (A) Schematic representation of the CHX trap in a hypoxia gradient. (B, C) To determine metastable p53 T dynamics in response to hypoxia, CHX trap design in (A) was used to capture p53 homo-oligomerization dynamics by anti-p53 BN-PAGE immune blotting at 1, 0.1 or 5% O 2 (immune blot is shown in Fig. 5B ). To sufficiently resolve each homo-oligomer (especially T and O) 5-15% Bis-tris gradient gel (pH 7.0) was utilized. T1 represents duration for which HCT116 p53+/+ cells were exposed to hypoxia before CHX treatment. Purple arrows indicate p53 pool segregated in its constituent homo-oligomers without CHX trap. T2 represents the duration of CHX for hypoxic cells. 24h > T2 > 6h was always maintained for p53T dynamics in 0-72h T1. A red arrow in (B) shows p53 aggregating smears. Native protein standards were run in the same gel and after transfer of samples on PVDF membrane; its lane was cut and stained separately with coomassie brilliant blue G250. Due to inclusion of protein standards in 15 well gel, 60 th h sample for 1% O 2 was analyzed separately or from other replicates. SDS-PAGE based analysis of total p53 pool and GAPDH loading control of immune blots in (B, C) is shown in Fig 5A, B or Fig S3F. (D) R.A. measurements from (B, C) show oxygen-sensitive p53T via shifts in equilibrium state (5% O 2 ). Green and magenta circles correspond to on-off pattern of p53 switch deciphered at 6h. The magenta arrow shows enhanced dimerization or octamerization via T during initial durations that initiates shifts at 1 and 0.1% O 2 respectively. Values and error bars in correspond to mean and standard deviation from three independent replicates of the experiment respectively and are best represented by the immune blots in (B, C) or Fig. 5B .

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, SDS Page, Standard Deviation

    p53 tetramer exists as the metastable state in basal conditions. (A) Schematic representation of the homo-oligomerization trap generated by CHX (100μM) and MG132. (B) Spontaneous p53 oscillations captured by the trap in the basal state of cells. (C) Anti-p53 BN-PAGE immune blot shows p53 homo-oligomerization in basal state of U2OS cells by −CHX (only MG132 intervention) or +CHX (CHX+MG132 interventions) variants of the trap. 3-17% Bis-tris gradient gel (pH 7.0) shows p53 M, D, T, O and H.O. forms. O is observed as diffused smears. The immune density of O smear shows enhancement with an increase in MG132 dose (μM) in −CHX or +CHX variations. NativeMark protein standards were cut from the PVDF membrane after protein transfer and stained separately with coomassie brilliant blue G250 (CBB) dye. (D) R.A. calculation was performed by the densitometry of immune blots that identifies D↽T (blue arrow) and T⇀O (magenta arrow) conversion as an indicator of metastability of p53 T through −CHX and +CHX trap variants in the basal state of the cells. Immune blot shown in (C) is the best representation of the data in (D). Values and error bars in (D) represent mean and standard deviation from three independent replicates of the experiment respectively.
    Figure Legend Snippet: p53 tetramer exists as the metastable state in basal conditions. (A) Schematic representation of the homo-oligomerization trap generated by CHX (100μM) and MG132. (B) Spontaneous p53 oscillations captured by the trap in the basal state of cells. (C) Anti-p53 BN-PAGE immune blot shows p53 homo-oligomerization in basal state of U2OS cells by −CHX (only MG132 intervention) or +CHX (CHX+MG132 interventions) variants of the trap. 3-17% Bis-tris gradient gel (pH 7.0) shows p53 M, D, T, O and H.O. forms. O is observed as diffused smears. The immune density of O smear shows enhancement with an increase in MG132 dose (μM) in −CHX or +CHX variations. NativeMark protein standards were cut from the PVDF membrane after protein transfer and stained separately with coomassie brilliant blue G250 (CBB) dye. (D) R.A. calculation was performed by the densitometry of immune blots that identifies D↽T (blue arrow) and T⇀O (magenta arrow) conversion as an indicator of metastability of p53 T through −CHX and +CHX trap variants in the basal state of the cells. Immune blot shown in (C) is the best representation of the data in (D). Values and error bars in (D) represent mean and standard deviation from three independent replicates of the experiment respectively.

    Techniques Used: Generated, Polyacrylamide Gel Electrophoresis, Staining, Standard Deviation

    2) Product Images from "The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria"

    Article Title: The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria

    Journal: The Plant Cell

    doi: 10.1105/tpc.16.00398

    Relative Accumulation of MatR and Various Mitochondrial Proteins during Arabidopsis Seed Germination. (A) ). Detection was performed by chemiluminescence with the Image Quant LAS4000 mini analyzer (GE Healthcare). The intensities of protein signals in (A) and (B) ). (B) -PAGE analysis of the respiratory chain complexes during seed germination in Arabidopsis. Crude membrane fractions obtained from dry seeds, imbibed seeds, and mature Arabidopsis seedlings were solubilized with DDM ( n ). For immunodetections, the proteins were transferred from the native gels onto a PVDF membrane (Bio-Rad) in cathode buffer for 15 h at 40 mA, using the Bio-Rad mini transblot cell. The membranes were distained with ethanol before probing with specific antibodies, as indicated below each blot. Arrows indicate the native respiratory complexes, CI (∼1000 kD), CIII (dimer, ∼500 kD), CIV (∼220 kD), and CV (∼600 kD), in Arabidopsis mitochondria. Please note, in (B) , the original COX2 blot has been modified, i.e., the lane corresponding of the mature leaves (M) was cut from the right side of the blot and pasted to the other side (marked with dotted line). No other changes have been made to the original figure.
    Figure Legend Snippet: Relative Accumulation of MatR and Various Mitochondrial Proteins during Arabidopsis Seed Germination. (A) ). Detection was performed by chemiluminescence with the Image Quant LAS4000 mini analyzer (GE Healthcare). The intensities of protein signals in (A) and (B) ). (B) -PAGE analysis of the respiratory chain complexes during seed germination in Arabidopsis. Crude membrane fractions obtained from dry seeds, imbibed seeds, and mature Arabidopsis seedlings were solubilized with DDM ( n ). For immunodetections, the proteins were transferred from the native gels onto a PVDF membrane (Bio-Rad) in cathode buffer for 15 h at 40 mA, using the Bio-Rad mini transblot cell. The membranes were distained with ethanol before probing with specific antibodies, as indicated below each blot. Arrows indicate the native respiratory complexes, CI (∼1000 kD), CIII (dimer, ∼500 kD), CIV (∼220 kD), and CV (∼600 kD), in Arabidopsis mitochondria. Please note, in (B) , the original COX2 blot has been modified, i.e., the lane corresponding of the mature leaves (M) was cut from the right side of the blot and pasted to the other side (marked with dotted line). No other changes have been made to the original figure.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification

    3) Product Images from "Expression Analysis of RNA-Binding Motif Gene on Y Chromosome (RBMY) Protein Isoforms in Testis Tissue and a Testicular Germ Cell Cancer-Derived Cell Line (NT2) "

    Article Title: Expression Analysis of RNA-Binding Motif Gene on Y Chromosome (RBMY) Protein Isoforms in Testis Tissue and a Testicular Germ Cell Cancer-Derived Cell Line (NT2)

    Journal: Iranian Biomedical Journal

    doi: 10.6091/ibj.1148.2013

    RBMY antibody confirmation by using Western-blot analysis of recombinant RBMY. Two concentrations (100 and 10 ng) of each recombinant protein were loaded on SDS-PAGE and after separation were transferred to PVDF membrane. Blots were exposed to X-ray films
    Figure Legend Snippet: RBMY antibody confirmation by using Western-blot analysis of recombinant RBMY. Two concentrations (100 and 10 ng) of each recombinant protein were loaded on SDS-PAGE and after separation were transferred to PVDF membrane. Blots were exposed to X-ray films

    Techniques Used: Western Blot, Recombinant, SDS Page

    4) Product Images from "Bivalent ligands incorporating curcumin and diosgenin as multifunctional compounds against Alzheimer’s disease"

    Article Title: Bivalent ligands incorporating curcumin and diosgenin as multifunctional compounds against Alzheimer’s disease

    Journal: Bioorganic & medicinal chemistry

    doi: 10.1016/j.bmc.2015.10.032

    Compounds 33 and 38 bind Aβ and reduce AβO formation, but have no effect on Aβ production. (A) Representative western blot. Cells were treated were lysed, and proteins were separated by SDS-PAGE. After transfer to a PVDF membrane, blots were probed with the 6E10 antibody. (B) Quantification of total Aβ oligomers from western blotting. Error bars represent SEM. (n=6; ** p
    Figure Legend Snippet: Compounds 33 and 38 bind Aβ and reduce AβO formation, but have no effect on Aβ production. (A) Representative western blot. Cells were treated were lysed, and proteins were separated by SDS-PAGE. After transfer to a PVDF membrane, blots were probed with the 6E10 antibody. (B) Quantification of total Aβ oligomers from western blotting. Error bars represent SEM. (n=6; ** p

    Techniques Used: Western Blot, SDS Page

    5) Product Images from "Control of organelle gene expression by the mitochondrial transcription termination factor mTERF22 in Arabidopsis thaliana plants"

    Article Title: Control of organelle gene expression by the mitochondrial transcription termination factor mTERF22 in Arabidopsis thaliana plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201631

    Relative accumulation of organellar proteins in wild-type and mterf22 plants. (A) Immunoblot analyses of wild-type plants and mterf22-1 mutant line. For the quantification of the relative abundances of organellar proteins in mterf22 plants, different amounts of total mitochondrial proteins extracted from wild-type plants were loaded and separated by SDS-PAGE. The blots were probed with polyclonal antibodies raised to different organellar proteins, as indicated in each panel. Detection was carried out by chemiluminescence assays after incubation with HRP-conjugated secondary antibody. (B) BN-PAGE of crude mitochondria preparations was performed according to the method described in [ 57 ]. Crude mitochondria preparations, obtained from 3-week-old Arabidopsis seedlings, were solubilized with DDM [1.5% (w/v)] and the organellar complexes were resolved by BN-PAGE. For immunodetection, proteins were transferred from the native gels onto a PVDF membrane and were probed with specific antibodies ( S2 Table ), as indicated below each blot. Arrows indicate to the native complexes I (~1,000 kDa), III (dimer, ~500 kDa), IV (~220 kDa) and V (~600 kDa). The asterisk in the CA2 panel indicates to the presence of a 700 ~ 800 kDa band, which may corresponds to a complex I assembly intermediate. Hybridization signals were analyzed by chemiluminescence assays after incubation with HRP-conjugated secondary antibody. The intensities of protein signals in panels ‘A’ and ‘B’ using ImageJ software [ 90 ].
    Figure Legend Snippet: Relative accumulation of organellar proteins in wild-type and mterf22 plants. (A) Immunoblot analyses of wild-type plants and mterf22-1 mutant line. For the quantification of the relative abundances of organellar proteins in mterf22 plants, different amounts of total mitochondrial proteins extracted from wild-type plants were loaded and separated by SDS-PAGE. The blots were probed with polyclonal antibodies raised to different organellar proteins, as indicated in each panel. Detection was carried out by chemiluminescence assays after incubation with HRP-conjugated secondary antibody. (B) BN-PAGE of crude mitochondria preparations was performed according to the method described in [ 57 ]. Crude mitochondria preparations, obtained from 3-week-old Arabidopsis seedlings, were solubilized with DDM [1.5% (w/v)] and the organellar complexes were resolved by BN-PAGE. For immunodetection, proteins were transferred from the native gels onto a PVDF membrane and were probed with specific antibodies ( S2 Table ), as indicated below each blot. Arrows indicate to the native complexes I (~1,000 kDa), III (dimer, ~500 kDa), IV (~220 kDa) and V (~600 kDa). The asterisk in the CA2 panel indicates to the presence of a 700 ~ 800 kDa band, which may corresponds to a complex I assembly intermediate. Hybridization signals were analyzed by chemiluminescence assays after incubation with HRP-conjugated secondary antibody. The intensities of protein signals in panels ‘A’ and ‘B’ using ImageJ software [ 90 ].

    Techniques Used: Mutagenesis, SDS Page, Incubation, Polyacrylamide Gel Electrophoresis, Immunodetection, Hybridization, Software

    6) Product Images from "Mouse Model for the Equilibration Interaction between the Host Immune System and Human T-Cell Leukemia Virus Type 1 Gene Expression"

    Article Title: Mouse Model for the Equilibration Interaction between the Host Immune System and Human T-Cell Leukemia Virus Type 1 Gene Expression

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.6.2703-2713.2002

    (A) Design of retrovirus vectors. The parental vector, pRTaxbsr, has a cytomegalovirus enhancer/promoter unit (CMV p) at the 5′ end, cDNA of Tax linked with a drug resistance gene, bsr , by an IRES, and an MLV LTR at the 3′ end. The 273-bp U3 region of MLV LTR was replaced by a 266-bp fragment of HTLV-1 LTR, resulting in pR3Taxbsr. Then cDNA of EGFP was fused with tax cDNA at the 5′ end, resulting in pR3Gaxbsr. (B) The protein expression of the pR and pR3 vectors was compared by Western blotting using an anti-Tax rabbit antiserum. BOSC23 cells were transfected with either pRTaxbsr or pR3Taxbsr by using Lipofectamine. NIH 3T3 cells were infected with the same titer of a supernatant of transfected BOSC23 cells. Proteins were harvested 50 h posttransfection and 48 h postinfection. Three micrograms of transfected BOSC23 cell lysates (lanes 1 and 2) and 17 μg of infected NIH 3T3 cell lysates (lanes 3 and 4) were analyzed by SDS-12% PAGE, blotted onto PVDF membranes, and then probed with a rabbit antiserum against the C terminus of Tax. (C) Establishment a mouse lymphoma cell line expressing Gax. EL4 cells were infected with pR3Gaxbsr virus, selected with Blasticidin S, cloned by limiting dilution, and then sorted as to EGFP intensity. The Gax-expressing cell line was established and named EL4/Gax. We also established EL4/Tax and EL4/EGFP cell lines by infection with RTaxbsr and REGFP viruses, respectively (data not shown).
    Figure Legend Snippet: (A) Design of retrovirus vectors. The parental vector, pRTaxbsr, has a cytomegalovirus enhancer/promoter unit (CMV p) at the 5′ end, cDNA of Tax linked with a drug resistance gene, bsr , by an IRES, and an MLV LTR at the 3′ end. The 273-bp U3 region of MLV LTR was replaced by a 266-bp fragment of HTLV-1 LTR, resulting in pR3Taxbsr. Then cDNA of EGFP was fused with tax cDNA at the 5′ end, resulting in pR3Gaxbsr. (B) The protein expression of the pR and pR3 vectors was compared by Western blotting using an anti-Tax rabbit antiserum. BOSC23 cells were transfected with either pRTaxbsr or pR3Taxbsr by using Lipofectamine. NIH 3T3 cells were infected with the same titer of a supernatant of transfected BOSC23 cells. Proteins were harvested 50 h posttransfection and 48 h postinfection. Three micrograms of transfected BOSC23 cell lysates (lanes 1 and 2) and 17 μg of infected NIH 3T3 cell lysates (lanes 3 and 4) were analyzed by SDS-12% PAGE, blotted onto PVDF membranes, and then probed with a rabbit antiserum against the C terminus of Tax. (C) Establishment a mouse lymphoma cell line expressing Gax. EL4 cells were infected with pR3Gaxbsr virus, selected with Blasticidin S, cloned by limiting dilution, and then sorted as to EGFP intensity. The Gax-expressing cell line was established and named EL4/Gax. We also established EL4/Tax and EL4/EGFP cell lines by infection with RTaxbsr and REGFP viruses, respectively (data not shown).

    Techniques Used: Plasmid Preparation, Expressing, Western Blot, Transfection, Infection, Polyacrylamide Gel Electrophoresis, Clone Assay

    7) Product Images from "Inflammatory breast cancer: Activation of the aryl hydrocarbon receptor and its target CYP1B1 correlates closely with Wnt5a/b-β-catenin signalling, the stem cell phenotype and disease progression"

    Article Title: Inflammatory breast cancer: Activation of the aryl hydrocarbon receptor and its target CYP1B1 correlates closely with Wnt5a/b-β-catenin signalling, the stem cell phenotype and disease progression

    Journal: Journal of Advanced Research

    doi: 10.1016/j.jare.2018.11.006

    Wnt5a/b and β-catenin expression in carcinoma tissues from non-IBC and IBC patients and their correlations with AHR/CYP1B1 and patents clinical pathological properties. (A) Representatives of protein expression of Wnt5a/b and β-catenin in non-IBC and IBC carcinoma tissue lysates analyzed by SDS-PAGE, transferred into PVDF membranes showing high expression of Wnt5a/b and β-catenin in breast carcinoma tissues of IBC patients. (B) Bars represent the relative density values of protein bands assessed by ImageJ software and normalized against the loading control β-tubulin and showing significant expression of Wnt5a/b and β-catenin in IBC tissues (n = 28) compared to non-IBC tissues (n = 33). Data represent the mean ± SD, * P ≤ 0.05 and ** P ≤ 0.001 as determined by Student’s t -test. (C and D) Scatter charts showing no correlation between AHR and CYP1B1 expression and Wnt5a/b and β-catenin expression in non-IBC carcinoma tissues. (E and F) Scatter charts showing a linear positive correlation between AHR/CYP1B1 expression and Wnt5a/b and β-catenin expression in IBC carcinoma tissues. (G and H) Bars represent expression of Wnt5a/b and β-catenin in non-IBC and IBC patients sub-grouped according to the tumour grade, both correlates with tumour grade in the non-IBC (G) and IBC (H) patient groups. (I and J) Bars represent the relative density values of Wnt5a/b and β-catenin proteins sub-grouped into negative and positive LVI, results showed no statistical correlation with LVI in non-IBC patients and a significant correlation with lymphovascular invasion in IBC patients. Data represent mean ± SD, * P ≤ 0.05 and ** P ≤ 0.001 as determined by one-way ANOVA followed by Tukey’s multiple comparison test. (K – N) Scatter charts showing a weak correlation between the number of metastatic lymph nodes and the relative density values of Wnt5a/b (K) and β-catenin (L) proteins in non-IBC tissues and a linear positive correlation between the number of metastatic lymph nodes and the relative density values of Wnt5a/b (M) and β-catenin (N) proteins in IBC tissues. Correlation coefficient (r values) were calculated by Pearson’s correlation test. (O and P) Bars represent the fold change (RQ = 2 −ΔΔCt ) of mRNA expression of Wnt5a in MDA-MB-231 (O) and SUM149 (P) cells after treatment with an AHR inhibitor. The results are representative of at least three independent experiments. Data represent the mean ± SD. * P ≤ 0.05 and ** P ≤ 0.001 as determined by Student’s t test.
    Figure Legend Snippet: Wnt5a/b and β-catenin expression in carcinoma tissues from non-IBC and IBC patients and their correlations with AHR/CYP1B1 and patents clinical pathological properties. (A) Representatives of protein expression of Wnt5a/b and β-catenin in non-IBC and IBC carcinoma tissue lysates analyzed by SDS-PAGE, transferred into PVDF membranes showing high expression of Wnt5a/b and β-catenin in breast carcinoma tissues of IBC patients. (B) Bars represent the relative density values of protein bands assessed by ImageJ software and normalized against the loading control β-tubulin and showing significant expression of Wnt5a/b and β-catenin in IBC tissues (n = 28) compared to non-IBC tissues (n = 33). Data represent the mean ± SD, * P ≤ 0.05 and ** P ≤ 0.001 as determined by Student’s t -test. (C and D) Scatter charts showing no correlation between AHR and CYP1B1 expression and Wnt5a/b and β-catenin expression in non-IBC carcinoma tissues. (E and F) Scatter charts showing a linear positive correlation between AHR/CYP1B1 expression and Wnt5a/b and β-catenin expression in IBC carcinoma tissues. (G and H) Bars represent expression of Wnt5a/b and β-catenin in non-IBC and IBC patients sub-grouped according to the tumour grade, both correlates with tumour grade in the non-IBC (G) and IBC (H) patient groups. (I and J) Bars represent the relative density values of Wnt5a/b and β-catenin proteins sub-grouped into negative and positive LVI, results showed no statistical correlation with LVI in non-IBC patients and a significant correlation with lymphovascular invasion in IBC patients. Data represent mean ± SD, * P ≤ 0.05 and ** P ≤ 0.001 as determined by one-way ANOVA followed by Tukey’s multiple comparison test. (K – N) Scatter charts showing a weak correlation between the number of metastatic lymph nodes and the relative density values of Wnt5a/b (K) and β-catenin (L) proteins in non-IBC tissues and a linear positive correlation between the number of metastatic lymph nodes and the relative density values of Wnt5a/b (M) and β-catenin (N) proteins in IBC tissues. Correlation coefficient (r values) were calculated by Pearson’s correlation test. (O and P) Bars represent the fold change (RQ = 2 −ΔΔCt ) of mRNA expression of Wnt5a in MDA-MB-231 (O) and SUM149 (P) cells after treatment with an AHR inhibitor. The results are representative of at least three independent experiments. Data represent the mean ± SD. * P ≤ 0.05 and ** P ≤ 0.001 as determined by Student’s t test.

    Techniques Used: Expressing, SDS Page, Software, Multiple Displacement Amplification

    mRNA and protein expression levels of AHR and CYP1B1 in non-IBC and IBC carcinoma tissues. Bars represent the fold change (RQ = 2 −ΔΔCt ) of mRNA expression of AHR and CYP1B1 in (A) breast cancer tissues (n = 61) after normalization to values of healthy tissues (n = 14), and in (B) IBC carcinoma tissues (n = 28) after normalization to values of non-IBC carcinoma tissues (n = 33). Data represent mean ± SD, * P ≤ 0.05 and ** P ≤ 0.001 as determined by Student’s t -test. (C) Representative of immunoblots membranes showing the protein expression of AHR and CYP1B1 in non-IBC and IBC, tissue lysates of non-IBC and IBC were analyzed by SDS-PAGE, transferred into PVDF membranes and immunoblotted with antibodies specific for AHR and CYP1B1. (D) Bars represent the relative density values of detected protein bands assessed by ImageJ software and normalized against the loading control β-tubulin, showing significantly higher expression of AHR and CYP1B1 in IBC (n = 28) than in non-IBC (n = 33) carcinoma tissues. (E) Representative fields of immunostaining (brown colour stain) of AHR and CYP1B1 in paraffin embedded breast carcinoma tissue sections showing high density of cancer cells positive for AHR and CYP1B1 in IBC (n = 20) compared to non-IBC patients (n = 25) (magnification 40X). (F) Bars represent the relative area fraction to healthy breast tissues calculated by using ImageJ software. Data represent the mean ± SD, * P ≤ 0.05 and ** P ≤ 0.001 as determined by Student’s t -test. (G and H) Represent scatter charts showing the positive correlations between the mRNA expression of AHR and CYP1B1 in non-IBC and IBC carcinoma tissues. (I and J) Represent scatter charts showing the positive correlations between the protein expression of AHR and CYP1B1 in non-IBC tissues and IBC tissues. Correlation coefficients (r values) were calculated by Pearson’s correlation test.
    Figure Legend Snippet: mRNA and protein expression levels of AHR and CYP1B1 in non-IBC and IBC carcinoma tissues. Bars represent the fold change (RQ = 2 −ΔΔCt ) of mRNA expression of AHR and CYP1B1 in (A) breast cancer tissues (n = 61) after normalization to values of healthy tissues (n = 14), and in (B) IBC carcinoma tissues (n = 28) after normalization to values of non-IBC carcinoma tissues (n = 33). Data represent mean ± SD, * P ≤ 0.05 and ** P ≤ 0.001 as determined by Student’s t -test. (C) Representative of immunoblots membranes showing the protein expression of AHR and CYP1B1 in non-IBC and IBC, tissue lysates of non-IBC and IBC were analyzed by SDS-PAGE, transferred into PVDF membranes and immunoblotted with antibodies specific for AHR and CYP1B1. (D) Bars represent the relative density values of detected protein bands assessed by ImageJ software and normalized against the loading control β-tubulin, showing significantly higher expression of AHR and CYP1B1 in IBC (n = 28) than in non-IBC (n = 33) carcinoma tissues. (E) Representative fields of immunostaining (brown colour stain) of AHR and CYP1B1 in paraffin embedded breast carcinoma tissue sections showing high density of cancer cells positive for AHR and CYP1B1 in IBC (n = 20) compared to non-IBC patients (n = 25) (magnification 40X). (F) Bars represent the relative area fraction to healthy breast tissues calculated by using ImageJ software. Data represent the mean ± SD, * P ≤ 0.05 and ** P ≤ 0.001 as determined by Student’s t -test. (G and H) Represent scatter charts showing the positive correlations between the mRNA expression of AHR and CYP1B1 in non-IBC and IBC carcinoma tissues. (I and J) Represent scatter charts showing the positive correlations between the protein expression of AHR and CYP1B1 in non-IBC tissues and IBC tissues. Correlation coefficients (r values) were calculated by Pearson’s correlation test.

    Techniques Used: Expressing, Western Blot, SDS Page, Software, Immunostaining, Staining

    8) Product Images from "Loss of MICOS complex integrity and mitochondrial damage, but not TDP-43 mitochondrial localisation, are likely associated with severity ofCHCHD10-related diseases"

    Article Title: Loss of MICOS complex integrity and mitochondrial damage, but not TDP-43 mitochondrial localisation, are likely associated with severity ofCHCHD10-related diseases

    Journal: Neurobiology of disease

    doi: 10.1016/j.nbd.2018.07.027

    Analysis of muscle and fibroblasts from patients bearing the p.Gly66Val mutation. A ). C: control individual. B . BN-PAGE of the MICOS and OXPHOS complexes in control (C) and patient (P6, P7) fibroblasts. Patients P6 and P7 carry the p.Gly66Val mutation. Complexes I to IV (CI-CIV) of OXPHOS were detected with an anti-NDUFS9 antibody (CI), an anti-SDHA antibody (CII), an anti-core I antibody (CIII) and an anti-cytochrome c oxidase subunit I antibody (CIV). MICOS and MIB complexes were detected with an antibody anti-MIC60/mitofilin. MIB : Mitochondrial Intermembrane space Bridging complex. C . Second dimension of the BN-PAGE showing that the steady-state levels of assembled CHCHD10 in MICOS complex are not affected in patient fibroblasts. D . Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN-PAGE from isolated mitochondria permeabilized with 6 g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC, supercomplexes I+III2+IVn. E . Representative western blot of MICOS proteins, including mitofilin (MIC60), CHCHD6 (MIC25) and CHCHD3 (MIC19) performed with fibroblast lysates obtained from controls (C1, C2) and patients (P6, P7). Three isoforms of mitofilin (89, 87 and 80kD) exist due to alternative splicing and can be detected by immunoblot analysis. HSP60 was used as a loading control. F .
    Figure Legend Snippet: Analysis of muscle and fibroblasts from patients bearing the p.Gly66Val mutation. A ). C: control individual. B . BN-PAGE of the MICOS and OXPHOS complexes in control (C) and patient (P6, P7) fibroblasts. Patients P6 and P7 carry the p.Gly66Val mutation. Complexes I to IV (CI-CIV) of OXPHOS were detected with an anti-NDUFS9 antibody (CI), an anti-SDHA antibody (CII), an anti-core I antibody (CIII) and an anti-cytochrome c oxidase subunit I antibody (CIV). MICOS and MIB complexes were detected with an antibody anti-MIC60/mitofilin. MIB : Mitochondrial Intermembrane space Bridging complex. C . Second dimension of the BN-PAGE showing that the steady-state levels of assembled CHCHD10 in MICOS complex are not affected in patient fibroblasts. D . Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN-PAGE from isolated mitochondria permeabilized with 6 g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC, supercomplexes I+III2+IVn. E . Representative western blot of MICOS proteins, including mitofilin (MIC60), CHCHD6 (MIC25) and CHCHD3 (MIC19) performed with fibroblast lysates obtained from controls (C1, C2) and patients (P6, P7). Three isoforms of mitofilin (89, 87 and 80kD) exist due to alternative splicing and can be detected by immunoblot analysis. HSP60 was used as a loading control. F .

    Techniques Used: Mutagenesis, Polyacrylamide Gel Electrophoresis, Isolation, Incubation, Western Blot

    9) Product Images from "RING3 Kinase Transactivates Promoters of Cell Cycle Regulatory Genes through E2F 1"

    Article Title: RING3 Kinase Transactivates Promoters of Cell Cycle Regulatory Genes through E2F 1

    Journal: Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research

    doi:

    Nuclear complexes that contain E2Fs interact with RING3. A , diagram of COOH-terminal deletions of RING3 N-tagged with six histidines (Δ Sna BI, Δ Bsp MI, Δ Bal I, Δ Hin dIII, Δ Pst I, and Δ Acc I), with the number of amino acids in RING3 shown on a scale ( above ). B , input HeLa nuclear extract ( Lane 1 ) and eluted proteins ( Lanes 2 and 3 ) from a nonimmune rabbit IgG affinity column ( Lane 2 ) or α RING3 rabbit IgG affinity column ( Lane 3 ) were separated by SDS-PAGE, electroblotted to PVDF, and probed with rabbit polyclonal antibody specific for all E2Fs ( arrow ). C , HeLa nuclear proteins were eluted from Ni-NTA columns charged with wild-type recombinant RING3 protein or with equal moles of the deletion mutants listed in A . Eluted fractions were immunoblotted with anti-E2F-1 or anti-E2F-2.
    Figure Legend Snippet: Nuclear complexes that contain E2Fs interact with RING3. A , diagram of COOH-terminal deletions of RING3 N-tagged with six histidines (Δ Sna BI, Δ Bsp MI, Δ Bal I, Δ Hin dIII, Δ Pst I, and Δ Acc I), with the number of amino acids in RING3 shown on a scale ( above ). B , input HeLa nuclear extract ( Lane 1 ) and eluted proteins ( Lanes 2 and 3 ) from a nonimmune rabbit IgG affinity column ( Lane 2 ) or α RING3 rabbit IgG affinity column ( Lane 3 ) were separated by SDS-PAGE, electroblotted to PVDF, and probed with rabbit polyclonal antibody specific for all E2Fs ( arrow ). C , HeLa nuclear proteins were eluted from Ni-NTA columns charged with wild-type recombinant RING3 protein or with equal moles of the deletion mutants listed in A . Eluted fractions were immunoblotted with anti-E2F-1 or anti-E2F-2.

    Techniques Used: Affinity Column, SDS Page, Recombinant

    10) Product Images from "Effects of rigidity on the selectivity of protein kinase inhibitors"

    Article Title: Effects of rigidity on the selectivity of protein kinase inhibitors

    Journal: European journal of medicinal chemistry

    doi: 10.1016/j.ejmech.2018.01.053

    Effects of vemurafenib and compound 2d on downstream signaling of BRAF V600E in A375 cells. Vemurafenib (VEM) potently blocked phosphorylation of MEK while 2d had little effects at up to 100 μM. A375 cells were treated with vemurafenib or compound 2d at various concentrations before the cells were lysed, resolved by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies for MEK and phospho-MEK (pMEK).
    Figure Legend Snippet: Effects of vemurafenib and compound 2d on downstream signaling of BRAF V600E in A375 cells. Vemurafenib (VEM) potently blocked phosphorylation of MEK while 2d had little effects at up to 100 μM. A375 cells were treated with vemurafenib or compound 2d at various concentrations before the cells were lysed, resolved by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies for MEK and phospho-MEK (pMEK).

    Techniques Used: SDS Page

    11) Product Images from "The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O‑Glucose"

    Article Title: The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O‑Glucose

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.7b13488

    A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O -GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β -actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β -elimination. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H 2 O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H 2 O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).
    Figure Legend Snippet: A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O -GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β -actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β -elimination. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H 2 O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H 2 O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).

    Techniques Used: Labeling, Western Blot, Negative Control, SDS Page, Incubation, Fluorescence

    12) Product Images from "The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O‑Glucose"

    Article Title: The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O‑Glucose

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.7b13488

    A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O -GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β -actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β -elimination. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H 2 O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H 2 O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).
    Figure Legend Snippet: A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O -GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β -actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β -elimination. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H 2 O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H 2 O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).

    Techniques Used: Labeling, Western Blot, Negative Control, SDS Page, Incubation, Fluorescence

    13) Product Images from "Anti-inflammatory and anti-cancer activity of mulberry (Morus alba L.) root bark"

    Article Title: Anti-inflammatory and anti-cancer activity of mulberry (Morus alba L.) root bark

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-14-200

    Effect of MRBE on IκB-α degradation (A), p65 nuclear translocation (B) and ERK1/2 phosphorylation (C) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-treated with MRBE at the indicated concentrations for 2 h and then co-treated with (1 μg/ml) for 15 min (for Western blot of IκB-α and ERK1/2 phosphorylation) or 30 min (for Western blot of p65). DMSO was used as a vehicle. Cell lysate were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies against IκB-α, p-ERK1/2, total ERK1/2 and p65. The proteins were then visualized using ECL detection. Actin was used as an internal control.
    Figure Legend Snippet: Effect of MRBE on IκB-α degradation (A), p65 nuclear translocation (B) and ERK1/2 phosphorylation (C) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-treated with MRBE at the indicated concentrations for 2 h and then co-treated with (1 μg/ml) for 15 min (for Western blot of IκB-α and ERK1/2 phosphorylation) or 30 min (for Western blot of p65). DMSO was used as a vehicle. Cell lysate were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies against IκB-α, p-ERK1/2, total ERK1/2 and p65. The proteins were then visualized using ECL detection. Actin was used as an internal control.

    Techniques Used: Translocation Assay, Western Blot, SDS Page

    Effect of MRBE on NO production (A) and iNOS (B) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-treated with MRBE at the indicated concentrations for 2 h and then co-treated with LPS (1 μg/ml) for the additional 18 h. After treatment, NO production was measured using the media and Griess reagent and cell lysates were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with iNOS antibody for Western blot. iNOS protein was visualized using ECL detection. Actin was used as internal control. DMSO was used as a vehicle. Values given are the mean ± SD (n = 3). *p
    Figure Legend Snippet: Effect of MRBE on NO production (A) and iNOS (B) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-treated with MRBE at the indicated concentrations for 2 h and then co-treated with LPS (1 μg/ml) for the additional 18 h. After treatment, NO production was measured using the media and Griess reagent and cell lysates were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with iNOS antibody for Western blot. iNOS protein was visualized using ECL detection. Actin was used as internal control. DMSO was used as a vehicle. Values given are the mean ± SD (n = 3). *p

    Techniques Used: SDS Page, Western Blot

    14) Product Images from "Function of translationally controlled tumor protein (TCTP) in Eudrilus eugeniae regeneration"

    Article Title: Function of translationally controlled tumor protein (TCTP) in Eudrilus eugeniae regeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175319

    Morphology of E . eugeniae and identification of E . eugeniae TCTP. (A) The earthworm, Eudrilus eugeniae , ar-anterior region, cl-clitellum, pr- posterior region. (B) The full length cDNA sequence of E . eugeniae TCTP ( tpt1 ) gene contain 504 bp. (C) Multiple sequence alignment of E . eugeniae TCTP protein sequence with its homologues of other animals using Align X of Vector NTI. It shows higher similarity of TCTP with the other animals including vertebrates. (D) Phylogenetic tree of E . eugeniae TCTP protein sequence with its homologues of other animals using Align X of Vector NTI. (E) Expression studies of E . eugeniae TCTP protein. The protein is 19 kDa in molecular weight. The 3 rd day regenerated tissue sample was analysed using immunoblot with anti-TCTP antibody. (F) Immunoblot with pre immune sera. It didn’t show any signals in PVDF membrane. (G) Immunoblot performed using anti- TCTP antibody with TCTP specific peptide. The peptide of TCTP which was used to raise the antibody, blocks the interact of the antibody with the TCTP molecules on the membrane. Each well was loaded with 40 μg of protein lyaste and the samples were resolved in 12% SDS-PAGE. (H) The expression of β-actin was used as a marker to confirm TCTP molecular weight.
    Figure Legend Snippet: Morphology of E . eugeniae and identification of E . eugeniae TCTP. (A) The earthworm, Eudrilus eugeniae , ar-anterior region, cl-clitellum, pr- posterior region. (B) The full length cDNA sequence of E . eugeniae TCTP ( tpt1 ) gene contain 504 bp. (C) Multiple sequence alignment of E . eugeniae TCTP protein sequence with its homologues of other animals using Align X of Vector NTI. It shows higher similarity of TCTP with the other animals including vertebrates. (D) Phylogenetic tree of E . eugeniae TCTP protein sequence with its homologues of other animals using Align X of Vector NTI. (E) Expression studies of E . eugeniae TCTP protein. The protein is 19 kDa in molecular weight. The 3 rd day regenerated tissue sample was analysed using immunoblot with anti-TCTP antibody. (F) Immunoblot with pre immune sera. It didn’t show any signals in PVDF membrane. (G) Immunoblot performed using anti- TCTP antibody with TCTP specific peptide. The peptide of TCTP which was used to raise the antibody, blocks the interact of the antibody with the TCTP molecules on the membrane. Each well was loaded with 40 μg of protein lyaste and the samples were resolved in 12% SDS-PAGE. (H) The expression of β-actin was used as a marker to confirm TCTP molecular weight.

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Molecular Weight, SDS Page, Marker

    15) Product Images from "The Actinobacillus pleuropneumoniae HMW1C-Like Glycosyltransferase Mediates N-Linked Glycosylation of the Haemophilus influenzae HMW1 Adhesin"

    Article Title: The Actinobacillus pleuropneumoniae HMW1C-Like Glycosyltransferase Mediates N-Linked Glycosylation of the Haemophilus influenzae HMW1 Adhesin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015888

    Specificity of HMW1ct glycosylation. The glycosylation reactions were carried out in standard conditions using His-HMW1ct as acceptor protein and UDP-glucose or UDP-galactose as donor substrate. ( A ) At each time point, an aliquot of the reaction was stopped by adding an equal volume of 2X SDS-PAGE sample buffer and followed by heating at 95°C for 4 min. ( B ) At each time point, SDS-PAGE samples were prepared as in A. However, two hrs after reaction with the first donor substrate, the second donor substrate was added to the reaction, as indicated. All samples were separated by 12% SDS-PAGE, and the gel was stained with Coomassie blue. The distinct shifts due to incorporated sugars are indicated by symbols (•, 0 hexose; ▪, 2 hexoses; ⋆, 4 or 5 hexoses; and ⋆’, 5 or 6 hexoses). ( C ) The glycosylation reactions were carried out in standard conditions using double mutants of His-HMW1ct (N1348Q/N1352Q, N1348Q/N1366Q, and N1352Q/N1366Q) by ApHMW1C using UDP-glucose or UDP-galactose as donor substrates. C1, C2, and C3 indicate control reactions without ApHMW1C. Samples were separated by 12% SDS-PAGE and were stained with Coomassie blue. ( D ) In parallel, a duplicated gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). The glycosylated proteins by UDP-glucose or by UDP-galactose are indicated by arrows. ( E ) Model of hexose modifications at Asn-1348, Asn-1352, and Asn-1366.
    Figure Legend Snippet: Specificity of HMW1ct glycosylation. The glycosylation reactions were carried out in standard conditions using His-HMW1ct as acceptor protein and UDP-glucose or UDP-galactose as donor substrate. ( A ) At each time point, an aliquot of the reaction was stopped by adding an equal volume of 2X SDS-PAGE sample buffer and followed by heating at 95°C for 4 min. ( B ) At each time point, SDS-PAGE samples were prepared as in A. However, two hrs after reaction with the first donor substrate, the second donor substrate was added to the reaction, as indicated. All samples were separated by 12% SDS-PAGE, and the gel was stained with Coomassie blue. The distinct shifts due to incorporated sugars are indicated by symbols (•, 0 hexose; ▪, 2 hexoses; ⋆, 4 or 5 hexoses; and ⋆’, 5 or 6 hexoses). ( C ) The glycosylation reactions were carried out in standard conditions using double mutants of His-HMW1ct (N1348Q/N1352Q, N1348Q/N1366Q, and N1352Q/N1366Q) by ApHMW1C using UDP-glucose or UDP-galactose as donor substrates. C1, C2, and C3 indicate control reactions without ApHMW1C. Samples were separated by 12% SDS-PAGE and were stained with Coomassie blue. ( D ) In parallel, a duplicated gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). The glycosylated proteins by UDP-glucose or by UDP-galactose are indicated by arrows. ( E ) Model of hexose modifications at Asn-1348, Asn-1352, and Asn-1366.

    Techniques Used: SDS Page, Staining

    Glycosylation of HMW1ct by ApHMW1C. To define the donor substrate specificity of ApHMW1C, glycosylation reactions were carried out in the reaction buffer with (R-lanes) or without (C-lanes) ApHMW1C using different UDP (or GDP) activated sugars. HMW1ct (without fusion tag) was used as the acceptor protein (lanes 1, and 3 to 6). As a control, His-tagged HMW1ct (His-HMW1ct) was also tested in a reaction with UDP-glucose as the donor sugar (lanes 2). ( A ) After the glycosylation reactions, samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue. ( B ) In parallel, a duplicate gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). Glycosylated HMW1ct proteins are indicated by arrows: ‘a’ and ‘c’ are glycosylated HMW1ct reacted with UDP-glucose and UDP-galactose, respectively, and ‘b’ is glycosylated His-HMW1ct reacted with UDP-glucose. The lanes labeled “M1,” “M2,” and “HMW1ct only” indicate pre-staining protein markers (Precision Plus Protein Standards, Bio-Rad), glycosylated protein markers (ProteoProfile PTM Marker, Sigma), and HMW1ct only as a control, respectively.
    Figure Legend Snippet: Glycosylation of HMW1ct by ApHMW1C. To define the donor substrate specificity of ApHMW1C, glycosylation reactions were carried out in the reaction buffer with (R-lanes) or without (C-lanes) ApHMW1C using different UDP (or GDP) activated sugars. HMW1ct (without fusion tag) was used as the acceptor protein (lanes 1, and 3 to 6). As a control, His-tagged HMW1ct (His-HMW1ct) was also tested in a reaction with UDP-glucose as the donor sugar (lanes 2). ( A ) After the glycosylation reactions, samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue. ( B ) In parallel, a duplicate gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). Glycosylated HMW1ct proteins are indicated by arrows: ‘a’ and ‘c’ are glycosylated HMW1ct reacted with UDP-glucose and UDP-galactose, respectively, and ‘b’ is glycosylated His-HMW1ct reacted with UDP-glucose. The lanes labeled “M1,” “M2,” and “HMW1ct only” indicate pre-staining protein markers (Precision Plus Protein Standards, Bio-Rad), glycosylated protein markers (ProteoProfile PTM Marker, Sigma), and HMW1ct only as a control, respectively.

    Techniques Used: SDS Page, Staining, Labeling, Marker

    N-linked glycosylation of HMW1ct. The glycosylation reactions were carried out in standard conditions using single (N1348Q, N1352Q, N1366Q) mutants of His-HMW1ct as acceptor proteins and UDP-glucose as donor substrate. ( A ) After the glycosylation reaction, the samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue. ( B ) A duplicate gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). The lanes labeled “Native” and “His-HMW1ct only” are control reaction samples with and without ApHMW1C, respectively. M1 is a pre-staining protein marker (Precision Plus Protein Standards, Bio-Rad), and M2 is a glycosylated protein marker (ProteoProfile PTM Marker, Sigma).
    Figure Legend Snippet: N-linked glycosylation of HMW1ct. The glycosylation reactions were carried out in standard conditions using single (N1348Q, N1352Q, N1366Q) mutants of His-HMW1ct as acceptor proteins and UDP-glucose as donor substrate. ( A ) After the glycosylation reaction, the samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue. ( B ) A duplicate gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). The lanes labeled “Native” and “His-HMW1ct only” are control reaction samples with and without ApHMW1C, respectively. M1 is a pre-staining protein marker (Precision Plus Protein Standards, Bio-Rad), and M2 is a glycosylated protein marker (ProteoProfile PTM Marker, Sigma).

    Techniques Used: SDS Page, Staining, Labeling, Marker

    16) Product Images from "Divergent effects of ?- and ?-myosin heavy chain isoforms on the N terminus of rat cardiac troponin T"

    Article Title: Divergent effects of ?- and ?-myosin heavy chain isoforms on the N terminus of rat cardiac troponin T

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201310971

    SDS-PAGE analysis of MHC isoform expression and Western blot analysis of reconstituted fibers. Solubilized muscle fiber samples from normal (α-MHC) and PTU-treated (β-MHC) rat hearts were run on a 6% SDS gel to estimate the isoform levels of MHC ( Ford and Chandra, 2013 ). (A) SDS gel showing the isoform expression of MHC in the left ventricles of normal and PTU-treated rats. Rats fed on a PTU diet show a near-complete shift to β-MHC isoform. (B) Western blot analysis of samples from reconstituted fibers expressing α-MHC. Reconstituted fibers were solubilized using 2% SDS ( Mamidi et al., 2012 ). Solubilized samples were run on an 8% SDS gel and transferred onto a PVDF membrane for Western blot analysis. Lane 1, purified recombinant RcTnT protein; lanes 2–4, samples from RcTnT WT -, RcTnT 1–43Δ -, and RcTnT 44–73Δ -reconstituted fibers. Molecular weights of proteins are indicated in parentheses.
    Figure Legend Snippet: SDS-PAGE analysis of MHC isoform expression and Western blot analysis of reconstituted fibers. Solubilized muscle fiber samples from normal (α-MHC) and PTU-treated (β-MHC) rat hearts were run on a 6% SDS gel to estimate the isoform levels of MHC ( Ford and Chandra, 2013 ). (A) SDS gel showing the isoform expression of MHC in the left ventricles of normal and PTU-treated rats. Rats fed on a PTU diet show a near-complete shift to β-MHC isoform. (B) Western blot analysis of samples from reconstituted fibers expressing α-MHC. Reconstituted fibers were solubilized using 2% SDS ( Mamidi et al., 2012 ). Solubilized samples were run on an 8% SDS gel and transferred onto a PVDF membrane for Western blot analysis. Lane 1, purified recombinant RcTnT protein; lanes 2–4, samples from RcTnT WT -, RcTnT 1–43Δ -, and RcTnT 44–73Δ -reconstituted fibers. Molecular weights of proteins are indicated in parentheses.

    Techniques Used: SDS Page, Expressing, Western Blot, SDS-Gel, Purification, Recombinant

    17) Product Images from "Molecular Cloning of a New Immunomodulatory Protein from Anoectochilus formosanus which Induces B Cell IgM Secretion through a T-Independent Mechanism"

    Article Title: Molecular Cloning of a New Immunomodulatory Protein from Anoectochilus formosanus which Induces B Cell IgM Secretion through a T-Independent Mechanism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021004

    Purification and biochemical characteristics of IPAF. (A) Crude protein extracts were fractionated by a DEAE-52 cellulose column eluted with linear gradient of 0.0–1.0 M NaCl. The elution profile was generated based on protein concentration determined by BCA analysis. (B) The active fractions were further purified using FPLC system equipped with a HiTrapQ anion exchange column eluted with linear gradient of 0.0–1.0 M NaCl. The elution profile was generated by measuring the absorbance at 280 nm. (C) Purified IPAF was identified by SDS-PAGE with Coomassie brilliant blue (lane 2) and periodic acid-Schiff staining (lane 3). Purified IPAF was transferred to PVDF membrane and identified via western blot using a self-made mAb against IPAF (lane 4). The molecular weight of IPAF was determined by comparing with pre-stained protein markers (lane 1). (D) Gel-filtration capillary electrophoresis SDS-MW analysis of IPAF prepared under reducing or non-reducing condition. The MW was derived by normalizing the migration time of the samples with the 10 kDa internal standard, and calibrated with the standard curve constructed with the protein size standard.
    Figure Legend Snippet: Purification and biochemical characteristics of IPAF. (A) Crude protein extracts were fractionated by a DEAE-52 cellulose column eluted with linear gradient of 0.0–1.0 M NaCl. The elution profile was generated based on protein concentration determined by BCA analysis. (B) The active fractions were further purified using FPLC system equipped with a HiTrapQ anion exchange column eluted with linear gradient of 0.0–1.0 M NaCl. The elution profile was generated by measuring the absorbance at 280 nm. (C) Purified IPAF was identified by SDS-PAGE with Coomassie brilliant blue (lane 2) and periodic acid-Schiff staining (lane 3). Purified IPAF was transferred to PVDF membrane and identified via western blot using a self-made mAb against IPAF (lane 4). The molecular weight of IPAF was determined by comparing with pre-stained protein markers (lane 1). (D) Gel-filtration capillary electrophoresis SDS-MW analysis of IPAF prepared under reducing or non-reducing condition. The MW was derived by normalizing the migration time of the samples with the 10 kDa internal standard, and calibrated with the standard curve constructed with the protein size standard.

    Techniques Used: Purification, Generated, Protein Concentration, BIA-KA, Fast Protein Liquid Chromatography, SDS Page, Staining, Western Blot, Molecular Weight, Filtration, Electrophoresis, Derivative Assay, Migration, Construct

    18) Product Images from "Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1"

    Article Title: Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1

    Journal: Cell Stress & Chaperones

    doi: 10.1007/s12192-017-0779-8

    J domain of Djp1 is partially dispensable for its peroxisomal protein import function. a Domain organization of Djp1. b Tenfold serial dilution of wild-type yeast cells harboring either an empty vector (−), CEN-ADH -Djp1 or CEN-TEF1 -Djp1 were spotted on selective media and incubated at indicated temperatures for 2 days. c Total protein lysate prepared from djp1Δ ) expressing empty vector ( djp1Δ ), wild-type cells harboring either empty vector (WT) or CEN-ADH- Djp1 [loading 0.5 OD 600 equivalent cells], and CEN-TEF1- Djp1 [loading 0.25 OD 600 equivalent cells] were resolved by SDS-PAGE, electro-blotted on PVDF membrane and probed with anti-Djp1 antibody (Djp1) (gift from Dr. B. Distel, Academic Medical Center, The Netherlands). A consistently appearing nonspecific band was used as a loading control ( c ) . d WT or djp1Δ cells harboring either empty CEN - ADH vector (−) or full-length Djp1, Djp1H34Q, Djp1 57–432 ( ΔJ ) expressed from a CEN-ADH ). Cells were visualized under fluorescence microscope at excitation/emission 470 nm/590 nm) (Zeiss, Apotome) at 100X. The scale bars indicate 1 μm. e Bar graph was created using ‘GraphPad Prism v. 5.00’ showing quantitative analysis for WT and djp1Δ cells with GFP-PTS1 harboring either wt Djp1 or its mutants. The experiments were done in triplicates and each time approximately 200 cells either having completely diffuse or punctate GFP fluorescence were manually counted using ImageJ software. Variations in the triplicates were depicted as error bars. f Expression level of Djp1 and its mutants wild-type (WT) or djp1Δ cells harboring either Djp1, ΔJ , H34Q expressed from a CEN - ADH -based plasmid was estimated by western analysis as mentioned above. g djp1Δ cells harboring either CEN - ADH -Djp1 or CEN - ADH -Djp1 1–282 ( ΔC50 ) plasmid was transformed with GFP-PTS1 and visualized under microscope as mentioned above. h Bar graph showing quantitative analysis for djp1Δ cells with GFP-PTS1 harboring either empty vector (−), Djp1 or Djp1( ΔC50 ) as mentioned above. i Western analysis of total protein lysate prepared from djp1Δ cells harboring either CEN - ADH -Djp1 or CEN - ADH -Djp1 ( ΔC50 ) plasmid was done as mentioned above
    Figure Legend Snippet: J domain of Djp1 is partially dispensable for its peroxisomal protein import function. a Domain organization of Djp1. b Tenfold serial dilution of wild-type yeast cells harboring either an empty vector (−), CEN-ADH -Djp1 or CEN-TEF1 -Djp1 were spotted on selective media and incubated at indicated temperatures for 2 days. c Total protein lysate prepared from djp1Δ ) expressing empty vector ( djp1Δ ), wild-type cells harboring either empty vector (WT) or CEN-ADH- Djp1 [loading 0.5 OD 600 equivalent cells], and CEN-TEF1- Djp1 [loading 0.25 OD 600 equivalent cells] were resolved by SDS-PAGE, electro-blotted on PVDF membrane and probed with anti-Djp1 antibody (Djp1) (gift from Dr. B. Distel, Academic Medical Center, The Netherlands). A consistently appearing nonspecific band was used as a loading control ( c ) . d WT or djp1Δ cells harboring either empty CEN - ADH vector (−) or full-length Djp1, Djp1H34Q, Djp1 57–432 ( ΔJ ) expressed from a CEN-ADH ). Cells were visualized under fluorescence microscope at excitation/emission 470 nm/590 nm) (Zeiss, Apotome) at 100X. The scale bars indicate 1 μm. e Bar graph was created using ‘GraphPad Prism v. 5.00’ showing quantitative analysis for WT and djp1Δ cells with GFP-PTS1 harboring either wt Djp1 or its mutants. The experiments were done in triplicates and each time approximately 200 cells either having completely diffuse or punctate GFP fluorescence were manually counted using ImageJ software. Variations in the triplicates were depicted as error bars. f Expression level of Djp1 and its mutants wild-type (WT) or djp1Δ cells harboring either Djp1, ΔJ , H34Q expressed from a CEN - ADH -based plasmid was estimated by western analysis as mentioned above. g djp1Δ cells harboring either CEN - ADH -Djp1 or CEN - ADH -Djp1 1–282 ( ΔC50 ) plasmid was transformed with GFP-PTS1 and visualized under microscope as mentioned above. h Bar graph showing quantitative analysis for djp1Δ cells with GFP-PTS1 harboring either empty vector (−), Djp1 or Djp1( ΔC50 ) as mentioned above. i Western analysis of total protein lysate prepared from djp1Δ cells harboring either CEN - ADH -Djp1 or CEN - ADH -Djp1 ( ΔC50 ) plasmid was done as mentioned above

    Techniques Used: Serial Dilution, Plasmid Preparation, Incubation, Expressing, SDS Page, Western Blot, Fluorescence, Microscopy, Software, Transformation Assay

    19) Product Images from "The Regulatory ? Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde-3-phosphate Dehydrogenase"

    Article Title: The Regulatory ? Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde-3-phosphate Dehydrogenase

    Journal: Biochemistry

    doi: 10.1021/bi800681g

    In situ phosphorylation in mouse skeletal muscle extracts. P/J (A) and I/lnJ (B) extracts were incubated either with Mg[γ- 32 P]ATP under different conditions [(a) without Ca 2+ and without exogenous PhK, (b) with Ca 2+ without exogenous PhK, and (c) with Ca 2+ and exogenous PhK] or without exogenous ATP (C). Reactions were carried out at 30 °C for 30 min and terminated by addition of UPPA-I buffer, the first component of the Perfect FOCUS kit purchased from Genotech Corp. (St. Louis, MO). Low-conductivity protein samples for isoelectric focusing were then prepared according to the manufacturer’s protocol. Proteins were resolved by 2D PAGE on linear gradient pH 3 to 10 strips (13 cm) from GE Healthcare (Uppsala, Sweden), followed by a second-dimension separation on a 5 to 17.5% T gradient gel. Gels were then stained, destained, dried, photographed, and exposed to a phosphor screen for 7–9 days. Phosphorylation patterns were visualized by autoradiography using an Amersham Biosciences Typhoon 9410 Imager and Image Quant verion 5.2 (Molecular Dynamics Corp., Sunnyvale, CA). Only the central part of the 16 cm × 18 cm gels is shown. A potential PhK target with a mass of ~42 kDa and a pI of ~6.6 (marked with an arrowhead within the gel image) was subjected to MALDI-MS analysis as described in Experimental Procedures. Proteins with similar masses and pI values of ~7.0, 7.6, 8.0, and 8.5 were also digested and analyzed by mass spectrometry. When no exogenous, radiolabeled ATP was used, proteins resolved by 2D PAGE were transferred to PVDF and stained with an anti- GAPDH polyclonal antibody (C).
    Figure Legend Snippet: In situ phosphorylation in mouse skeletal muscle extracts. P/J (A) and I/lnJ (B) extracts were incubated either with Mg[γ- 32 P]ATP under different conditions [(a) without Ca 2+ and without exogenous PhK, (b) with Ca 2+ without exogenous PhK, and (c) with Ca 2+ and exogenous PhK] or without exogenous ATP (C). Reactions were carried out at 30 °C for 30 min and terminated by addition of UPPA-I buffer, the first component of the Perfect FOCUS kit purchased from Genotech Corp. (St. Louis, MO). Low-conductivity protein samples for isoelectric focusing were then prepared according to the manufacturer’s protocol. Proteins were resolved by 2D PAGE on linear gradient pH 3 to 10 strips (13 cm) from GE Healthcare (Uppsala, Sweden), followed by a second-dimension separation on a 5 to 17.5% T gradient gel. Gels were then stained, destained, dried, photographed, and exposed to a phosphor screen for 7–9 days. Phosphorylation patterns were visualized by autoradiography using an Amersham Biosciences Typhoon 9410 Imager and Image Quant verion 5.2 (Molecular Dynamics Corp., Sunnyvale, CA). Only the central part of the 16 cm × 18 cm gels is shown. A potential PhK target with a mass of ~42 kDa and a pI of ~6.6 (marked with an arrowhead within the gel image) was subjected to MALDI-MS analysis as described in Experimental Procedures. Proteins with similar masses and pI values of ~7.0, 7.6, 8.0, and 8.5 were also digested and analyzed by mass spectrometry. When no exogenous, radiolabeled ATP was used, proteins resolved by 2D PAGE were transferred to PVDF and stained with an anti- GAPDH polyclonal antibody (C).

    Techniques Used: In Situ, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Autoradiography, Mass Spectrometry

    20) Product Images from "Establishment and Characterization of Monoclonal Antibodies Against SARS Coronavirus"

    Article Title: Establishment and Characterization of Monoclonal Antibodies Against SARS Coronavirus

    Journal: SARS- and Other Coronaviruses

    doi: 10.1007/978-1-59745-181-9_15

    Immunoblot of SARS-CoV proteins with monoclonal antibodies. Purified SARS-CoV proteins (0.5 μg/lane) are electrophoresed with SDS-PAGE (under reducing conditions), blotted to PVDF membrane, and detected by incubation with mAbs against SARS-CoV proteins. The detection is done with peroxidase-labeled-F(ab′) 2 anti-mouse IgG followed by chemiluminescent reaction: (A) Mouse serum from SARS-CoV immunized mouse. (B) Anti-N mAb, SKOT-8. (C) Anti-N mAb, SKOT-9. (D) Anti-S mAb, SOAT13. The positions of molecular weight markers are shown on the left. (Reproduced from( 6 ) with permission.)
    Figure Legend Snippet: Immunoblot of SARS-CoV proteins with monoclonal antibodies. Purified SARS-CoV proteins (0.5 μg/lane) are electrophoresed with SDS-PAGE (under reducing conditions), blotted to PVDF membrane, and detected by incubation with mAbs against SARS-CoV proteins. The detection is done with peroxidase-labeled-F(ab′) 2 anti-mouse IgG followed by chemiluminescent reaction: (A) Mouse serum from SARS-CoV immunized mouse. (B) Anti-N mAb, SKOT-8. (C) Anti-N mAb, SKOT-9. (D) Anti-S mAb, SOAT13. The positions of molecular weight markers are shown on the left. (Reproduced from( 6 ) with permission.)

    Techniques Used: Purification, SDS Page, Incubation, Labeling, Molecular Weight

    21) Product Images from "Bacillus anthracis’ lethal toxin induces broad transcriptional responses in human peripheral monocytes"

    Article Title: Bacillus anthracis’ lethal toxin induces broad transcriptional responses in human peripheral monocytes

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-13-33

    Monocyte purity, apoptosis, and susceptibility to LT. Red = CD14+ monocytes. Green = CD14-lymphocytes. A.) Forward and side scatter analysis of purified fixed human monocytes showing the monocyte population as compared to total population. B.) CD14 Pacific Blue and forward scatter analysis of fixed purified human monocytes showing > 85% monocytes. C.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h incubation showing 99.0% viable cells indicated in quadrant 3. D.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h LT treatment showing 99.1% viable cells indicated in quadrant 3. HeLa cells or human monocytes were left untreated or treated with 500 ng/mL LT for 4 h at 37°C. Samples were lysed, run on SDS-PAGE, transferred to PVDF membrane, and probed with indicated antibodies. Both MEK3 and MEK1 were cleaved by LT while control cells showed no MEK cleavage. β-actin loading controls show equivalent loading of both control and LT treated cells.
    Figure Legend Snippet: Monocyte purity, apoptosis, and susceptibility to LT. Red = CD14+ monocytes. Green = CD14-lymphocytes. A.) Forward and side scatter analysis of purified fixed human monocytes showing the monocyte population as compared to total population. B.) CD14 Pacific Blue and forward scatter analysis of fixed purified human monocytes showing > 85% monocytes. C.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h incubation showing 99.0% viable cells indicated in quadrant 3. D.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h LT treatment showing 99.1% viable cells indicated in quadrant 3. HeLa cells or human monocytes were left untreated or treated with 500 ng/mL LT for 4 h at 37°C. Samples were lysed, run on SDS-PAGE, transferred to PVDF membrane, and probed with indicated antibodies. Both MEK3 and MEK1 were cleaved by LT while control cells showed no MEK cleavage. β-actin loading controls show equivalent loading of both control and LT treated cells.

    Techniques Used: Purification, Incubation, SDS Page

    22) Product Images from "Sac7 and Rho1 regulate the white-to-opaque switching in Candida albicans"

    Article Title: Sac7 and Rho1 regulate the white-to-opaque switching in Candida albicans

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19246-9

    Sac7 physically interacts with Rho1. ( a ) Amino acid sequence alignment of Ca Rho1 with Sc Cdc42. Consensus residues are shaded. The arrowhead and asterisk indicate the conserved residues mutated to generate GTP- and GDP-locked forms of GTPase, respectively. ( b ) Sac7-HA interacts with Myc-Rho1 G18V but not Myc-Rho1 D124A . Lysates prepared from cells expressing Sac7-HA (YSL607), Myc-Rho1 G18V (YSL570), Myc-Rho1 D124A (YSL564), Sac7-HA and Myc-Rho1 G18V (YSL601), and Sac7-HA and Myc-Rho1 D124A (YSL609) were immunoprecipitated with the HA antibody. The immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membrane for WB analysis with HA and Myc antibodies. The cell lysates were also directly subjected to WB analysis with the Myc antibody as a control.
    Figure Legend Snippet: Sac7 physically interacts with Rho1. ( a ) Amino acid sequence alignment of Ca Rho1 with Sc Cdc42. Consensus residues are shaded. The arrowhead and asterisk indicate the conserved residues mutated to generate GTP- and GDP-locked forms of GTPase, respectively. ( b ) Sac7-HA interacts with Myc-Rho1 G18V but not Myc-Rho1 D124A . Lysates prepared from cells expressing Sac7-HA (YSL607), Myc-Rho1 G18V (YSL570), Myc-Rho1 D124A (YSL564), Sac7-HA and Myc-Rho1 G18V (YSL601), and Sac7-HA and Myc-Rho1 D124A (YSL609) were immunoprecipitated with the HA antibody. The immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membrane for WB analysis with HA and Myc antibodies. The cell lysates were also directly subjected to WB analysis with the Myc antibody as a control.

    Techniques Used: Sequencing, Expressing, Immunoprecipitation, SDS Page, Western Blot

    23) Product Images from "Mechanism of Metal Ion-Induced Activation of a Two-Component Sensor Kinase"

    Article Title: Mechanism of Metal Ion-Induced Activation of a Two-Component Sensor Kinase

    Journal: The Biochemical journal

    doi: 10.1042/BCJ20180577

    In vitro autophosphorylation of CusS cp wild-type and CusS cp -H271A. (A) Dot blot analysis of CusS cp . CusS cp was diluted to 10 μM in kinase buffer and autophosphorylation was initiated by adding ATP and incubated at 37°C. Samples were collected at 0 and 30 min, then 2 μL of each was spotted on PVDF membrane and probed with anti-1-pHis and anti-His tag antibodies simultaneously (left), or with anti-3-pHis and anti-His tag antibodies simultaneously (right). (B) CusS cp (25 μM) was diluted in kinase buffer containing ATP to a final concentration of 1 mM, and reactions were incubated at 37°C. Samples were collected at the indicated times (2 min to 2 hr). (C) CusS cp -H271A (25 μM) was diluted in kinase buffer containing ATP to a final concentration of 1 mM, and reactions were incubated at 37°C. Samples were collected at the indicated times (5 min to 1 hr). The reactions were terminated by adding 2X SDS sample buffer and were loaded onto SDS-PAGE. After electrophoresis, the proteins were transferred onto PVDF membranes and analyzed by Western blot probed with anti-1-pHis and anti-His-tag antibodies simultaneously. For all panels, each experiment was repeated at least three times; a representative experiment is shown.
    Figure Legend Snippet: In vitro autophosphorylation of CusS cp wild-type and CusS cp -H271A. (A) Dot blot analysis of CusS cp . CusS cp was diluted to 10 μM in kinase buffer and autophosphorylation was initiated by adding ATP and incubated at 37°C. Samples were collected at 0 and 30 min, then 2 μL of each was spotted on PVDF membrane and probed with anti-1-pHis and anti-His tag antibodies simultaneously (left), or with anti-3-pHis and anti-His tag antibodies simultaneously (right). (B) CusS cp (25 μM) was diluted in kinase buffer containing ATP to a final concentration of 1 mM, and reactions were incubated at 37°C. Samples were collected at the indicated times (2 min to 2 hr). (C) CusS cp -H271A (25 μM) was diluted in kinase buffer containing ATP to a final concentration of 1 mM, and reactions were incubated at 37°C. Samples were collected at the indicated times (5 min to 1 hr). The reactions were terminated by adding 2X SDS sample buffer and were loaded onto SDS-PAGE. After electrophoresis, the proteins were transferred onto PVDF membranes and analyzed by Western blot probed with anti-1-pHis and anti-His-tag antibodies simultaneously. For all panels, each experiment was repeated at least three times; a representative experiment is shown.

    Techniques Used: In Vitro, Dot Blot, Incubation, Concentration Assay, SDS Page, Electrophoresis, Western Blot

    24) Product Images from "The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX"

    Article Title: The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.00544

    Evaluation of the gG3PD enzymatic activity. (A) Spectrophotometric analysis of purified HIS-gG3PD. The UV-visible spectrum of HIS-gG3PD (10 mg/ml) in 67 mM of potassium phosphate buffer, pH 7.5, was recorded at 25°C. The insert shows a magnification of the HIS-gG3PD spectrum (solid line) in comparison with the spectrum of authentic FAD (dotted line) recorded in the same buffer. Peak maxima are reported. Spectra are representative of three independent experiments. (B) Assessment of HIS-gG3PD dimerization in vitro . Purified recombinant proteins (3 μmol) were separated on 3–12% Blue Native-PAGE and silver-stained or transferred on polyvinylidene difluoride (PVDF) membrane and probed with anti-HIS mAb. Native size markers (kDa) are indicated on the left. Asterisks indicate HIS-gG3PD monomer ( ∗ ) or dimer ( ∗∗ ). Empty dots indicate HIS-gG3PD_C monomer (°) or dimer ( °° ). The arrow indicates HIS-gG3PD_N monomer (
    Figure Legend Snippet: Evaluation of the gG3PD enzymatic activity. (A) Spectrophotometric analysis of purified HIS-gG3PD. The UV-visible spectrum of HIS-gG3PD (10 mg/ml) in 67 mM of potassium phosphate buffer, pH 7.5, was recorded at 25°C. The insert shows a magnification of the HIS-gG3PD spectrum (solid line) in comparison with the spectrum of authentic FAD (dotted line) recorded in the same buffer. Peak maxima are reported. Spectra are representative of three independent experiments. (B) Assessment of HIS-gG3PD dimerization in vitro . Purified recombinant proteins (3 μmol) were separated on 3–12% Blue Native-PAGE and silver-stained or transferred on polyvinylidene difluoride (PVDF) membrane and probed with anti-HIS mAb. Native size markers (kDa) are indicated on the left. Asterisks indicate HIS-gG3PD monomer ( ∗ ) or dimer ( ∗∗ ). Empty dots indicate HIS-gG3PD_C monomer (°) or dimer ( °° ). The arrow indicates HIS-gG3PD_N monomer (

    Techniques Used: Activity Assay, Purification, In Vitro, Recombinant, Blue Native PAGE, Staining

    25) Product Images from "Regulated release of ERdj3 from unfolded proteins by BiP"

    Article Title: Regulated release of ERdj3 from unfolded proteins by BiP

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2008.207

    WT and QPD ERdj3 bind to denatured luciferase similarly in vitro . ( A ) Temperature denatured (D) or native (N) luciferase (Luc) was directly loaded on a gel (first two lanes) or incubated with bacterially produced recombinant wild-type ERdj3 and then immunoprecipitated with either anti-ERdj3 polyclonal antiserum or with Protein A Sepharose beads alone. Reaction cocktails were subjected to reducing SDS–PAGE and then transferred to a PVDF membrane. The membrane was blotted with either goat anti-luciferase antiserum followed by donkey anti-goat Ig conjugated to HRP or with the polyclonal anti-ERdj3 followed by goat anti-rabbit Ig conjugated to HRP. In both cases, the signal was detected by chemiluminescence. ( B ) Chemically denatured luciferase (solid grey bars) or binding buffer alone (hatched bars) was used to coat 96-well plates. Recombinant wild-type or the QPD mutant ERdj3 proteins (0.5 μM) were added to the wells and bound ERdj3 was detected with a polyclonal anti-ERdj3 antiserum, followed by donkey anti-rabbit Ig conjugated to alkaline phosphatase. The DNTP substrate was added and after developing, the plates were read on a spectrophotometer and the signal was expressed in OD units. A luciferase-coated well that did not receive ERdj3 protein was treated similarly and serves as a negative control for the antibody (dark grey). All samples were run in triplicate and error bars are indicated.
    Figure Legend Snippet: WT and QPD ERdj3 bind to denatured luciferase similarly in vitro . ( A ) Temperature denatured (D) or native (N) luciferase (Luc) was directly loaded on a gel (first two lanes) or incubated with bacterially produced recombinant wild-type ERdj3 and then immunoprecipitated with either anti-ERdj3 polyclonal antiserum or with Protein A Sepharose beads alone. Reaction cocktails were subjected to reducing SDS–PAGE and then transferred to a PVDF membrane. The membrane was blotted with either goat anti-luciferase antiserum followed by donkey anti-goat Ig conjugated to HRP or with the polyclonal anti-ERdj3 followed by goat anti-rabbit Ig conjugated to HRP. In both cases, the signal was detected by chemiluminescence. ( B ) Chemically denatured luciferase (solid grey bars) or binding buffer alone (hatched bars) was used to coat 96-well plates. Recombinant wild-type or the QPD mutant ERdj3 proteins (0.5 μM) were added to the wells and bound ERdj3 was detected with a polyclonal anti-ERdj3 antiserum, followed by donkey anti-rabbit Ig conjugated to alkaline phosphatase. The DNTP substrate was added and after developing, the plates were read on a spectrophotometer and the signal was expressed in OD units. A luciferase-coated well that did not receive ERdj3 protein was treated similarly and serves as a negative control for the antibody (dark grey). All samples were run in triplicate and error bars are indicated.

    Techniques Used: Luciferase, In Vitro, Incubation, Produced, Recombinant, Immunoprecipitation, SDS Page, Binding Assay, Mutagenesis, Spectrophotometry, Negative Control

    26) Product Images from "Role of cell surface vimentin in Chandipura virus replication in Neuro-2a cells"

    Article Title: Role of cell surface vimentin in Chandipura virus replication in Neuro-2a cells

    Journal: Virus Research

    doi: 10.1016/j.virusres.2020.198014

    Western blot analysis to demonstrate the presence of vimentin in cytoplasmic and membrane extract. The Neuro-2a cell membrane and cytoplasmic proteins were resolved on SDS-PAGE, transferred to PVDF membrane and probed with anti-vimentin antibody. Note the presence of vimentin in cytoplasmic and membrane fractions.
    Figure Legend Snippet: Western blot analysis to demonstrate the presence of vimentin in cytoplasmic and membrane extract. The Neuro-2a cell membrane and cytoplasmic proteins were resolved on SDS-PAGE, transferred to PVDF membrane and probed with anti-vimentin antibody. Note the presence of vimentin in cytoplasmic and membrane fractions.

    Techniques Used: Western Blot, SDS Page

    Virus overlay protein binding assay (VOPBA). The membrane proteins from Neuro-2a cells were resolved on SDS-PAGE and transferred to PVDF membrane. The PEG precipitated CHPV was overlaid onto the membrane and interacting proteins was detected using anti-CHPV rabbit immune sera. A protein band of 55 kDa was seen in the CHPV overlaid membrane (a) while the corresponding band was absent in the control membrane (b). Protein sizes were indicated on left.
    Figure Legend Snippet: Virus overlay protein binding assay (VOPBA). The membrane proteins from Neuro-2a cells were resolved on SDS-PAGE and transferred to PVDF membrane. The PEG precipitated CHPV was overlaid onto the membrane and interacting proteins was detected using anti-CHPV rabbit immune sera. A protein band of 55 kDa was seen in the CHPV overlaid membrane (a) while the corresponding band was absent in the control membrane (b). Protein sizes were indicated on left.

    Techniques Used: Protein Binding, SDS Page

    27) Product Images from "Salmonella Heterogeneously Expresses Flagellin during Colonization of Plants"

    Article Title: Salmonella Heterogeneously Expresses Flagellin during Colonization of Plants

    Journal: Microorganisms

    doi: 10.3390/microorganisms8060815

    Flagellin synthesis is reduced in plant-based media. Presence of flagellin protein was determined using western blot ( a ) and ELISA ( b ) techniques. Total Salmonella proteins were extracted 24 or 48 h after inoculation of S. Typhimurium 14028s into LM, TM, MM or LB media. Inoculation with the S. Typhimurium ΔfljBΔfliC mutant was used as a negative control. Five µg of total protein was separated on SDS–PAGE and blotted on a PVDF membrane prior to probing with a primary anti- Salmonella specific flagellin ( anti-FliC ) antibody, followed by probing with secondary anti-mouse antibody coupled to HRP enzyme and exposition ( a ). Alternatively, 96-well plates were coated with proteins isolated as indicated and probed with primary ( anti-FliC ) and secondary anti-mouse antibody coupled to HRP antibodies. The resulting substrate production was assessed 30 min after reaction start using 450 nm and 652 nm wavelengths. As internal control, several dilutions of the wild type strain growing in LB at 24 hpi were used to compare the expression level. The dilutions used were 10 8 , 10 5 , and 10 4 CFU/mL ( b ).
    Figure Legend Snippet: Flagellin synthesis is reduced in plant-based media. Presence of flagellin protein was determined using western blot ( a ) and ELISA ( b ) techniques. Total Salmonella proteins were extracted 24 or 48 h after inoculation of S. Typhimurium 14028s into LM, TM, MM or LB media. Inoculation with the S. Typhimurium ΔfljBΔfliC mutant was used as a negative control. Five µg of total protein was separated on SDS–PAGE and blotted on a PVDF membrane prior to probing with a primary anti- Salmonella specific flagellin ( anti-FliC ) antibody, followed by probing with secondary anti-mouse antibody coupled to HRP enzyme and exposition ( a ). Alternatively, 96-well plates were coated with proteins isolated as indicated and probed with primary ( anti-FliC ) and secondary anti-mouse antibody coupled to HRP antibodies. The resulting substrate production was assessed 30 min after reaction start using 450 nm and 652 nm wavelengths. As internal control, several dilutions of the wild type strain growing in LB at 24 hpi were used to compare the expression level. The dilutions used were 10 8 , 10 5 , and 10 4 CFU/mL ( b ).

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis, Negative Control, SDS Page, Isolation, Expressing

    28) Product Images from "Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools"

    Article Title: Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools

    Journal: The Prostate

    doi: 10.1002/pros.23311

    Epitope mapping and mAb Western blotting. (Panel A): Alignment of the epitopes on PSMA from different species recognized by the mAbs 1A11 and 3F11 as revealed by peptide scanning. Panel B: Purified ectodomains of human PSMA and several orthologs/paralogs, cell culture supernatants as well as cell lysates were separated by reducing 10% SDS–PAGE, electrotransferred onto a PVDF membrane, and probed with individual mAbs. Lanes: 1. human PSMA-overexpressing HEK293T/17 lysate (0.5 μg); 2. GCP3 overexpressing HEK293T/17 lysate (50 μg); 3. HEK293T/17 lysate (50 μg); 4. LNCaP lysate (30 μg); 5. CW22Rv1 lysate (30 μg); 6. PC-3 lysate (50 μg); 7. human PSMA (10 ng); 8. human PSMA (2 ng); 9. human GCP3 (40 ng); 10. human GCP3 (8 ng); 11. mouse PSMA (10 ng); 12. mouse GCP3 (cell culture supernatant; 15 μl); 13. Rat PSMA (cell culture supernatant; 1.5 μl); 14. pig PSMA (cell culture supernatant; 1 μl).
    Figure Legend Snippet: Epitope mapping and mAb Western blotting. (Panel A): Alignment of the epitopes on PSMA from different species recognized by the mAbs 1A11 and 3F11 as revealed by peptide scanning. Panel B: Purified ectodomains of human PSMA and several orthologs/paralogs, cell culture supernatants as well as cell lysates were separated by reducing 10% SDS–PAGE, electrotransferred onto a PVDF membrane, and probed with individual mAbs. Lanes: 1. human PSMA-overexpressing HEK293T/17 lysate (0.5 μg); 2. GCP3 overexpressing HEK293T/17 lysate (50 μg); 3. HEK293T/17 lysate (50 μg); 4. LNCaP lysate (30 μg); 5. CW22Rv1 lysate (30 μg); 6. PC-3 lysate (50 μg); 7. human PSMA (10 ng); 8. human PSMA (2 ng); 9. human GCP3 (40 ng); 10. human GCP3 (8 ng); 11. mouse PSMA (10 ng); 12. mouse GCP3 (cell culture supernatant; 15 μl); 13. Rat PSMA (cell culture supernatant; 1.5 μl); 14. pig PSMA (cell culture supernatant; 1 μl).

    Techniques Used: Western Blot, Purification, Cell Culture, SDS Page

    29) Product Images from "Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting"

    Article Title: Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting

    Journal: Journal of Biomolecular Techniques : JBT

    doi: 10.7171/jbt.16-2703-004

    Effects of reusing gel EB and fresh TTB on Western blots. A 25 μg CFBE-wt cell lysate was electrophoresed on individual, 4–20% Mini-PROTEAN TGX gels, transferred to a PVDF membrane, and subjected to Western blotting. Fresh SDS-PAGE buffer
    Figure Legend Snippet: Effects of reusing gel EB and fresh TTB on Western blots. A 25 μg CFBE-wt cell lysate was electrophoresed on individual, 4–20% Mini-PROTEAN TGX gels, transferred to a PVDF membrane, and subjected to Western blotting. Fresh SDS-PAGE buffer

    Techniques Used: Western Blot, SDS Page

    Effects of reusing gel EB and TTB on Western blots. A 25 μg CFBE-wt cell lysate was electrophoresed on individual, 4–20% Mini-PROTEAN TGX gels, as described in , transferred to PVDF membrane, and subjected to Western blotting. Fresh
    Figure Legend Snippet: Effects of reusing gel EB and TTB on Western blots. A 25 μg CFBE-wt cell lysate was electrophoresed on individual, 4–20% Mini-PROTEAN TGX gels, as described in , transferred to PVDF membrane, and subjected to Western blotting. Fresh

    Techniques Used: Western Blot

    30) Product Images from "Structure-Based Design and Engineering of a Nontoxic Recombinant Pokeweed Antiviral Protein with Potent Anti-Human Immunodeficiency Virus Activity"

    Article Title: Structure-Based Design and Engineering of a Nontoxic Recombinant Pokeweed Antiviral Protein with Potent Anti-Human Immunodeficiency Virus Activity

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.47.3.1052-1061.2003

    (A) Association of PAP mutants with ribosomes isolated from rabbit reticulocyte-enriched blood. (A.1) Total ribosomal protein (5 μg) was incubated with 1 μg of PAP and the ribosome-PAP complexes were isolated by ultracentrifugation. The ribosome-PAP complexes were separated through an SDS-12% polyacrylamide gel, electroblotted onto a PVDF membrane, and immunoblotted with a polyclonal antibody to PAP. (A.2) Prior to separation of the PAP-ribosome complex, a fraction (5 μl) of the reaction mixture was removed, separated through an SDS-12% polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with a polyclonal antibody to PAP. The results show that equal amounts of PAPs were added to the reaction mixture. (B) Association of wild-type and mutant PAPs with in vitro-synthesized ribosomal protein L3. (B.1) Coimmunoprecipitated PAP revealed by immunoblotting with anti-PAP antibody. 35 S-labeled L3 was incubated with wild-type and mutant PAPs and coimmunoprecipitated with protein A-Sepharose beads precoated with monoclonal antibody to L3. The PAP-L3 complexes were separated through SDS-12% polyacrylamide gels, transferred to a PVDF membrane, and immunoblotted with a polyclonal anti-PAP antibody. (B.2) The blot was exposed to X-ray film, which shows equal amounts of labeled L3 protein in each reaction mixture. (B.3) Prior to coimmunoprecipitation, a fraction (5 μl) was removed from the reaction mixture, separated through an SDS-12% polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with a polyclonal antibody to PAP. The results show that equal amounts of PAP were added to each reaction mixture.
    Figure Legend Snippet: (A) Association of PAP mutants with ribosomes isolated from rabbit reticulocyte-enriched blood. (A.1) Total ribosomal protein (5 μg) was incubated with 1 μg of PAP and the ribosome-PAP complexes were isolated by ultracentrifugation. The ribosome-PAP complexes were separated through an SDS-12% polyacrylamide gel, electroblotted onto a PVDF membrane, and immunoblotted with a polyclonal antibody to PAP. (A.2) Prior to separation of the PAP-ribosome complex, a fraction (5 μl) of the reaction mixture was removed, separated through an SDS-12% polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with a polyclonal antibody to PAP. The results show that equal amounts of PAPs were added to the reaction mixture. (B) Association of wild-type and mutant PAPs with in vitro-synthesized ribosomal protein L3. (B.1) Coimmunoprecipitated PAP revealed by immunoblotting with anti-PAP antibody. 35 S-labeled L3 was incubated with wild-type and mutant PAPs and coimmunoprecipitated with protein A-Sepharose beads precoated with monoclonal antibody to L3. The PAP-L3 complexes were separated through SDS-12% polyacrylamide gels, transferred to a PVDF membrane, and immunoblotted with a polyclonal anti-PAP antibody. (B.2) The blot was exposed to X-ray film, which shows equal amounts of labeled L3 protein in each reaction mixture. (B.3) Prior to coimmunoprecipitation, a fraction (5 μl) was removed from the reaction mixture, separated through an SDS-12% polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with a polyclonal antibody to PAP. The results show that equal amounts of PAP were added to each reaction mixture.

    Techniques Used: Isolation, Incubation, Papanicolaou Stain, Mutagenesis, In Vitro, Synthesized, Labeling

    31) Product Images from "ANTI-11[E]-PYROGLUTAMATE-MODIFIED AMYLOID ? ANTIBODIES CROSS-REACT WITH OTHER PATHOLOGICAL A? SPECIES: RELEVANCE FOR IMMUNOTHERAPY"

    Article Title: ANTI-11[E]-PYROGLUTAMATE-MODIFIED AMYLOID ? ANTIBODIES CROSS-REACT WITH OTHER PATHOLOGICAL A? SPECIES: RELEVANCE FOR IMMUNOTHERAPY

    Journal: Journal of neuroimmunology

    doi: 10.1016/j.jneuroim.2010.08.020

    Dot blot analysis. Aβ monomers were applied to PVDF membrane as shown in A) and detected with rabbit anti-Aβ1-42 (B), anti-AβN3(pE) (C) and anti-AβN11(pE) (D) antibodies.
    Figure Legend Snippet: Dot blot analysis. Aβ monomers were applied to PVDF membrane as shown in A) and detected with rabbit anti-Aβ1-42 (B), anti-AβN3(pE) (C) and anti-AβN11(pE) (D) antibodies.

    Techniques Used: Dot Blot

    32) Product Images from "The C-terminal tail of the NEIL1 DNA glycosylase interacts with the human mitochondrial single-stranded DNA binding protein"

    Article Title: The C-terminal tail of the NEIL1 DNA glycosylase interacts with the human mitochondrial single-stranded DNA binding protein

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2018.02.012

    Residues within the C-terminal tail of NEIL1 interact with mtSSB. (A) Domain organization of full-length (FL) His-tagged NEIL1, C-terminal deletion constructs, and GST-tagged C-terminal constructs of the NEIL1 DNA glycosylase. (B) Far-western analysis to determine the minimal construct of NEIL1 required for an interaction with mtSSB. All constructs used in this study were expressed in E. coli , purified to homogeneity and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis stained with Coomassie blue (left panel) . 50 pmol of all NEIL1 constructs, bovine serum albumin (negative control), glutathione S-transferase (negative control), and 10 pmol of mtSSB were loaded onto the gel. Far-western analysis was performed where proteins were transferred to a PVDF membrane, denatured, slowly renatured on the membrane, and incubated with either 10 pmol/ml purified mtSSB (middle panel) or 1 mg/ml HCT-116 whole cell extract (right panel) , and probed with an anti-mtSSB antibody to detect an interaction.
    Figure Legend Snippet: Residues within the C-terminal tail of NEIL1 interact with mtSSB. (A) Domain organization of full-length (FL) His-tagged NEIL1, C-terminal deletion constructs, and GST-tagged C-terminal constructs of the NEIL1 DNA glycosylase. (B) Far-western analysis to determine the minimal construct of NEIL1 required for an interaction with mtSSB. All constructs used in this study were expressed in E. coli , purified to homogeneity and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis stained with Coomassie blue (left panel) . 50 pmol of all NEIL1 constructs, bovine serum albumin (negative control), glutathione S-transferase (negative control), and 10 pmol of mtSSB were loaded onto the gel. Far-western analysis was performed where proteins were transferred to a PVDF membrane, denatured, slowly renatured on the membrane, and incubated with either 10 pmol/ml purified mtSSB (middle panel) or 1 mg/ml HCT-116 whole cell extract (right panel) , and probed with an anti-mtSSB antibody to detect an interaction.

    Techniques Used: Construct, Western Blot, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Negative Control, Incubation

    33) Product Images from "Cultured bloodstream Trypanosoma brucei adapt to life without mitochondrial translation release factor 1"

    Article Title: Cultured bloodstream Trypanosoma brucei adapt to life without mitochondrial translation release factor 1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23472-6

    Loss of TbMrf1 affects the structural integrity of protein complexes that contain mt encoded subunits. ( a ) The sedimentation pattern of 12S and 9S rRNAs in BF 427 and dKO TbMrf1 1wk and 7wk cell lines. Whole cell lysates from 5 × 10 8 parasites were resolved on a 10–30% glycerol gradient. RNA was extracted from each fraction and separated on 5% polyacrylamide/8 M urea gels that were blotted and probed for 12S and 9S mitochondrial rRNAs and 18S cytosolic rRNA (sedimentation control). BF 427–8 μg of total RNA from BF 427. Input – 3 μg of total RNA isolated from the remaining material that was loaded on a gradient. ( b ) After normalization to BF 427 RNA, the relative intensities of 9S and 12S rRNA signals from each sample were plotted. ( c ) The native F 1 - and F o F 1 -ATPase complexes were visualized using light blue native electrophoresis. Purified mitochondria from BF 427 and dKO TbMrf1 1wk and 7wk cultures were lysed with dodecyl maltoside, fractionated on 3–12% BisTris gel and blotted on a PVDF membrane. The F 1 -ATPase (F 1 ) and the F o F 1 -ATPase monomer and dimer were all visualized using specific polyclonal antibodies against F 1 -ATPase subunit β and F o -ATPase subunit OSCP. ( d ) SDS-PAGE Western blot analyses of the same mitochondrial lysates as in ( c ). The steady state abundance of mt hsp70, TbAAC, F 1 -ATPase subunits β and p18 and F o -ATPase subunits OSCP and ATPaseTb2 were determined using specific antibodies.
    Figure Legend Snippet: Loss of TbMrf1 affects the structural integrity of protein complexes that contain mt encoded subunits. ( a ) The sedimentation pattern of 12S and 9S rRNAs in BF 427 and dKO TbMrf1 1wk and 7wk cell lines. Whole cell lysates from 5 × 10 8 parasites were resolved on a 10–30% glycerol gradient. RNA was extracted from each fraction and separated on 5% polyacrylamide/8 M urea gels that were blotted and probed for 12S and 9S mitochondrial rRNAs and 18S cytosolic rRNA (sedimentation control). BF 427–8 μg of total RNA from BF 427. Input – 3 μg of total RNA isolated from the remaining material that was loaded on a gradient. ( b ) After normalization to BF 427 RNA, the relative intensities of 9S and 12S rRNA signals from each sample were plotted. ( c ) The native F 1 - and F o F 1 -ATPase complexes were visualized using light blue native electrophoresis. Purified mitochondria from BF 427 and dKO TbMrf1 1wk and 7wk cultures were lysed with dodecyl maltoside, fractionated on 3–12% BisTris gel and blotted on a PVDF membrane. The F 1 -ATPase (F 1 ) and the F o F 1 -ATPase monomer and dimer were all visualized using specific polyclonal antibodies against F 1 -ATPase subunit β and F o -ATPase subunit OSCP. ( d ) SDS-PAGE Western blot analyses of the same mitochondrial lysates as in ( c ). The steady state abundance of mt hsp70, TbAAC, F 1 -ATPase subunits β and p18 and F o -ATPase subunits OSCP and ATPaseTb2 were determined using specific antibodies.

    Techniques Used: Sedimentation, Isolation, Electrophoresis, Purification, SDS Page, Western Blot

    34) Product Images from "Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection"

    Article Title: Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2008.0792

    Expression and purification of EnvC from 143B cells infected with rVV–GFP–envC. ( A–B ) Infected cells were solubilized in Laemmli buffer and fractionated in gradient PAGE gel (4–12%); proteins were transferred to the PVDF membrane and probed with ( A ) anti-HIV-1 human IgG isolated from serum of an HIV-1–infected patient and ( B ) normal human IgG. ( C–D ) Analysis of purified mutant HIV envelope glycoproteins produced in 143B cells infected with rVV–GFP–envC. The mutant HIV EnvC yielded the expected band of approximately 140 kDa under denaturing conditions. ( C ) SDS-PAGE Coomassie blue staining. Lane 1, 10 μg of total protein from mock-infected cells; lane 2, 10 μg of protein purified by lentil lectin affinity column and Superdex-S200 size exclusion column chromatography using extracts of cells infected with rVV–GFP–envC. ( D ) Silver staining. Lane 1, total protein (2 μg) after the lentil lectin affinity column purification; lane 2, purified protein (2 μg) after size exclusion column chromatography purification.
    Figure Legend Snippet: Expression and purification of EnvC from 143B cells infected with rVV–GFP–envC. ( A–B ) Infected cells were solubilized in Laemmli buffer and fractionated in gradient PAGE gel (4–12%); proteins were transferred to the PVDF membrane and probed with ( A ) anti-HIV-1 human IgG isolated from serum of an HIV-1–infected patient and ( B ) normal human IgG. ( C–D ) Analysis of purified mutant HIV envelope glycoproteins produced in 143B cells infected with rVV–GFP–envC. The mutant HIV EnvC yielded the expected band of approximately 140 kDa under denaturing conditions. ( C ) SDS-PAGE Coomassie blue staining. Lane 1, 10 μg of total protein from mock-infected cells; lane 2, 10 μg of protein purified by lentil lectin affinity column and Superdex-S200 size exclusion column chromatography using extracts of cells infected with rVV–GFP–envC. ( D ) Silver staining. Lane 1, total protein (2 μg) after the lentil lectin affinity column purification; lane 2, purified protein (2 μg) after size exclusion column chromatography purification.

    Techniques Used: Expressing, Purification, Infection, Polyacrylamide Gel Electrophoresis, Isolation, Mutagenesis, Produced, SDS Page, Staining, Affinity Column, Column Chromatography, Silver Staining

    35) Product Images from "Generation of a transgenic zebrafish model of Tauopathy using a novel promoter element derived from the zebrafish eno2 gene"

    Article Title: Generation of a transgenic zebrafish model of Tauopathy using a novel promoter element derived from the zebrafish eno2 gene

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm608

    Expression of 4R-Tau in the brains of adult Tg( eno2:Tau ) zebrafish. ( A ) Ten micrograms of protein derived from wild-type AB* zebrafish brain, normal human post-mortem cerebral cortex or Tg( eno2:Tau ) Pt406 zebrafish brain was separated by SDS–PAGE and transferred to a PVDF membrane. The resulting western blot was probed sequentially with an antibody specific to human Tau (upper panel) and an antibody that binds to both human and zebrafish actin (lower panel). ( B ) Sections from wild-type AB* (upper two panels) or Tg( eno2:Tau ) Pt406 (lower two panels) zebrafish brains were immunolabelled using the human Tau-specific antibody shown in (A). Bound antibody was detected using a peroxidase-conjugated secondary antibody and a histochemical reaction with a red reaction product, as shown in Figure 5 A. Nuclei were counterstained blue. The left two panels show low-power views of the optic tectum to demonstrate the regional pattern of Tau expression in axons and neuropil. The right two panels show high-power views of the thalamus to demonstrate expression of Tau in the cell bodies and proximal processes of neurons, which are identified by their large pale-staining nuclei.
    Figure Legend Snippet: Expression of 4R-Tau in the brains of adult Tg( eno2:Tau ) zebrafish. ( A ) Ten micrograms of protein derived from wild-type AB* zebrafish brain, normal human post-mortem cerebral cortex or Tg( eno2:Tau ) Pt406 zebrafish brain was separated by SDS–PAGE and transferred to a PVDF membrane. The resulting western blot was probed sequentially with an antibody specific to human Tau (upper panel) and an antibody that binds to both human and zebrafish actin (lower panel). ( B ) Sections from wild-type AB* (upper two panels) or Tg( eno2:Tau ) Pt406 (lower two panels) zebrafish brains were immunolabelled using the human Tau-specific antibody shown in (A). Bound antibody was detected using a peroxidase-conjugated secondary antibody and a histochemical reaction with a red reaction product, as shown in Figure 5 A. Nuclei were counterstained blue. The left two panels show low-power views of the optic tectum to demonstrate the regional pattern of Tau expression in axons and neuropil. The right two panels show high-power views of the thalamus to demonstrate expression of Tau in the cell bodies and proximal processes of neurons, which are identified by their large pale-staining nuclei.

    Techniques Used: Expressing, Derivative Assay, SDS Page, Western Blot, Staining

    36) Product Images from "Cation-chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes"

    Article Title: Cation-chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200105026

    Topo II directly binds Ca 2 + , which inhibits the catalytic activity. (A) Topo II relaxation activity inhibited by Ca 2 + in vitro. Supercoiled plasmid DNA and 1 U of purified human Topo II in the presence of different Mg 2 +/Ca 2 + ratios were incubated for 5–20 min at 30°C. The inlay shows a 1.5% agarose gel of the relaxation experiments with supercoiled (FI) and relaxed (FII) forms after incubation with Topo II. Note the 1:3 Mg 2 +/Ca 2 + ratio detected at metaphase chromosomes using SIMS reduced the Topo II relaxation activity > 90%. (B) Direct detection of Ca 2 +-binding proteins: SDS gradient gels were stained with Coomassie blue (C) (M, marker, 1,3, and 6) or transferred to PVDF membranes, incubated with 1 mM CaCl 2 and then with 1 mM quin-2 (Q) and photographed after illumination with UV light (M, 2,4, and 7) or incubated with Topo II antibodies (5) or with Topo II, hCAP-C and ScII-specific antibodies (8) in Western analysis (W). Protein marker (M) (Sigma-Aldrich) represents 0.35 μg of each protein: rabbit myosin (205 kD), E. coli galactosidase (116 kD), rabbit phosphorylase b (97.4 kD), bovine albumin (66 kD), egg albumin (45 kD), and bovine carbonic anhydrase (29 kD). As a positive control, we used 0.75-μg bovine calmodulin (Sigma-Aldrich) (17 kD) (1 and 2). (Lanes 3,4, and 5) 5 U of purified human Topo II (TopoGen) are shown. Lanes 6, 7, and 8 represent purified fractionated IM chromosomes.
    Figure Legend Snippet: Topo II directly binds Ca 2 + , which inhibits the catalytic activity. (A) Topo II relaxation activity inhibited by Ca 2 + in vitro. Supercoiled plasmid DNA and 1 U of purified human Topo II in the presence of different Mg 2 +/Ca 2 + ratios were incubated for 5–20 min at 30°C. The inlay shows a 1.5% agarose gel of the relaxation experiments with supercoiled (FI) and relaxed (FII) forms after incubation with Topo II. Note the 1:3 Mg 2 +/Ca 2 + ratio detected at metaphase chromosomes using SIMS reduced the Topo II relaxation activity > 90%. (B) Direct detection of Ca 2 +-binding proteins: SDS gradient gels were stained with Coomassie blue (C) (M, marker, 1,3, and 6) or transferred to PVDF membranes, incubated with 1 mM CaCl 2 and then with 1 mM quin-2 (Q) and photographed after illumination with UV light (M, 2,4, and 7) or incubated with Topo II antibodies (5) or with Topo II, hCAP-C and ScII-specific antibodies (8) in Western analysis (W). Protein marker (M) (Sigma-Aldrich) represents 0.35 μg of each protein: rabbit myosin (205 kD), E. coli galactosidase (116 kD), rabbit phosphorylase b (97.4 kD), bovine albumin (66 kD), egg albumin (45 kD), and bovine carbonic anhydrase (29 kD). As a positive control, we used 0.75-μg bovine calmodulin (Sigma-Aldrich) (17 kD) (1 and 2). (Lanes 3,4, and 5) 5 U of purified human Topo II (TopoGen) are shown. Lanes 6, 7, and 8 represent purified fractionated IM chromosomes.

    Techniques Used: Activity Assay, In Vitro, Plasmid Preparation, Purification, Incubation, Agarose Gel Electrophoresis, Binding Assay, Staining, Marker, Western Blot, Positive Control

    37) Product Images from "SmCL3, a Gastrodermal Cysteine Protease of the Human Blood Fluke Schistosoma mansoni"

    Article Title: SmCL3, a Gastrodermal Cysteine Protease of the Human Blood Fluke Schistosoma mansoni

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0000449

    Detection of SmCL3 by Western blot in soluble S. mansoni extracts using mouse polyclonal IgG antibodies. Recombinant enzyme or soluble S. mansoni protein extracts (each 20 µg) were resolved by SDS-PAGE (15%) and electroblotted onto PVDF membrane. IgG purified antibodies reacted with (1) deglycosylated recombinant protein, (2) glycosylated protein, soluble extracts of (3) adults, (4) eggs and (7) 1 day old in vitro transformed schistosomula. Extracts of (5) miracidia and (6) cercariae did not react.
    Figure Legend Snippet: Detection of SmCL3 by Western blot in soluble S. mansoni extracts using mouse polyclonal IgG antibodies. Recombinant enzyme or soluble S. mansoni protein extracts (each 20 µg) were resolved by SDS-PAGE (15%) and electroblotted onto PVDF membrane. IgG purified antibodies reacted with (1) deglycosylated recombinant protein, (2) glycosylated protein, soluble extracts of (3) adults, (4) eggs and (7) 1 day old in vitro transformed schistosomula. Extracts of (5) miracidia and (6) cercariae did not react.

    Techniques Used: Western Blot, Recombinant, SDS Page, Purification, In Vitro, Transformation Assay

    38) Product Images from "Tracking the amphibian pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans using a highly specific monoclonal antibody and lateral‐flow technology"

    Article Title: Tracking the amphibian pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans using a highly specific monoclonal antibody and lateral‐flow technology

    Journal: Microbial Biotechnology

    doi: 10.1111/1751-7915.12464

    Immunodetection of 5C4 antigen in amphibian tissue samples. (A) Western immunoblot of soluble antigens in swabs of amphibian tissues naturally infected with Bd and Bsal . Soluble antigens present in swabs of foot, pelvic or skin fragments were subjected to denaturing SDS ‐ PAGE and transferred electrophoretically to a PVDF membrane. The membrane was probed with tissue culture supernatant of 5C4 followed by goat anti‐mouse IgM (μ‐chain‐specific) alkaline phosphatase conjugate and BCIP / NBT substrate. The antibody reacted with soluble antigens present in tissue swabs from frogs (samples 3, 6, 7, 10, 11 and 12) and newts and salamanders (samples 24 and 25) naturally infected with Batrachochytrium dendrobatidis ( Bd ) and Batrachochytrium salamandrivorans ( Bsal ) respectively. The 5C4 antibody was able to differentiate between Bd and Bsal , giving two distinct patterns of antigen binding. The 5C4‐negative sample 23 from a Southern Marbled newt ( Triturus pygmaeus ) was similarly negative in ELISA tests with 5C4, but was positive by Bsal qPCR and strongly positive with the 5C4 lateral‐flow assay (Table 4 ). The 5C4‐positive sample 3 from an Australian green tree frog ( Litoria caerulea ), which was negative by Bd qPCR (Table 4 ), was positive in ELISA and LFA tests and, in histology, this animal was shown to have a chytrid‐like infection. (B) Strong positive LFA test result for swab from tissue sample 23 ( Bsal qPCR positive, Western blot negative and ELISA negative), weak positive LFA test result for sample 7 ( Bd qPCR positive, Western blot positive and ELISA positive with 5C4) and negative LFA test result for sample 33 (negative by Bd qPCR , negative by Bsal qPCR and negative by Western blot and ELISA with 5C4).
    Figure Legend Snippet: Immunodetection of 5C4 antigen in amphibian tissue samples. (A) Western immunoblot of soluble antigens in swabs of amphibian tissues naturally infected with Bd and Bsal . Soluble antigens present in swabs of foot, pelvic or skin fragments were subjected to denaturing SDS ‐ PAGE and transferred electrophoretically to a PVDF membrane. The membrane was probed with tissue culture supernatant of 5C4 followed by goat anti‐mouse IgM (μ‐chain‐specific) alkaline phosphatase conjugate and BCIP / NBT substrate. The antibody reacted with soluble antigens present in tissue swabs from frogs (samples 3, 6, 7, 10, 11 and 12) and newts and salamanders (samples 24 and 25) naturally infected with Batrachochytrium dendrobatidis ( Bd ) and Batrachochytrium salamandrivorans ( Bsal ) respectively. The 5C4 antibody was able to differentiate between Bd and Bsal , giving two distinct patterns of antigen binding. The 5C4‐negative sample 23 from a Southern Marbled newt ( Triturus pygmaeus ) was similarly negative in ELISA tests with 5C4, but was positive by Bsal qPCR and strongly positive with the 5C4 lateral‐flow assay (Table 4 ). The 5C4‐positive sample 3 from an Australian green tree frog ( Litoria caerulea ), which was negative by Bd qPCR (Table 4 ), was positive in ELISA and LFA tests and, in histology, this animal was shown to have a chytrid‐like infection. (B) Strong positive LFA test result for swab from tissue sample 23 ( Bsal qPCR positive, Western blot negative and ELISA negative), weak positive LFA test result for sample 7 ( Bd qPCR positive, Western blot positive and ELISA positive with 5C4) and negative LFA test result for sample 33 (negative by Bd qPCR , negative by Bsal qPCR and negative by Western blot and ELISA with 5C4).

    Techniques Used: Immunodetection, Western Blot, Infection, SDS Page, Binding Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Lateral Flow Assay

    Characterization of the 5C4 antigen and extracellular antigen production. (A) Western immunoblot with 5C4 using the Bd ‐ GPL JEL 423 immunogen and following treatment of PVDF membranes with acetate buffer only (lane 1) or with periodate (lane 2). The reduction of immunoreactivity of 5C4 with glycoproteins between ∼27 and ∼220 kDa following periodate treatment shows that the antibody binds to carbohydrate moieties containing vicinal hydroxyl groups. Wells were loaded with 1.6 μg protein. M r denotes molecular weight in kDa. (B) Western immunoblot with 5C4 using washed, lyophilized zoospores of Bd ‐ GPL JEL 423. Wells were loaded with 1.6 μg protein. M r denotes molecular weight in kDa. (C) ELISA absorbance values at 450 nm for extracellular 5C4‐reactive antigens present in liquid cultures of Bd ‐ GPL JEL 423. Each point is the mean of three biological ± standard errors. The increase in absorbance values over the 7‐day sampling period shows that the antigen is shed into the external environment during growth and differentiation of the fungus.
    Figure Legend Snippet: Characterization of the 5C4 antigen and extracellular antigen production. (A) Western immunoblot with 5C4 using the Bd ‐ GPL JEL 423 immunogen and following treatment of PVDF membranes with acetate buffer only (lane 1) or with periodate (lane 2). The reduction of immunoreactivity of 5C4 with glycoproteins between ∼27 and ∼220 kDa following periodate treatment shows that the antibody binds to carbohydrate moieties containing vicinal hydroxyl groups. Wells were loaded with 1.6 μg protein. M r denotes molecular weight in kDa. (B) Western immunoblot with 5C4 using washed, lyophilized zoospores of Bd ‐ GPL JEL 423. Wells were loaded with 1.6 μg protein. M r denotes molecular weight in kDa. (C) ELISA absorbance values at 450 nm for extracellular 5C4‐reactive antigens present in liquid cultures of Bd ‐ GPL JEL 423. Each point is the mean of three biological ± standard errors. The increase in absorbance values over the 7‐day sampling period shows that the antigen is shed into the external environment during growth and differentiation of the fungus.

    Techniques Used: Western Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay, Sampling

    39) Product Images from "Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection"

    Article Title: Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9799

    2D gel electrophoresis and western blot analysis of M. bovis HB0801 TX114 membrane fractions A. Resolution of the HB0801 TX114 membrane fraction using 2-DE. Isoelectric points, ranging from pH 3-10, are indicated at the top and molecular weight markers in kDa on the left. B. Antigenic pattern of the M. bovis HB0801 membrane fraction obtained using a serum pool from calves experimentally infected with HB0801. The 17 spots identified by MALDI-TOF MS are indicated on the 2-DE map and the PVDF membrane.
    Figure Legend Snippet: 2D gel electrophoresis and western blot analysis of M. bovis HB0801 TX114 membrane fractions A. Resolution of the HB0801 TX114 membrane fraction using 2-DE. Isoelectric points, ranging from pH 3-10, are indicated at the top and molecular weight markers in kDa on the left. B. Antigenic pattern of the M. bovis HB0801 membrane fraction obtained using a serum pool from calves experimentally infected with HB0801. The 17 spots identified by MALDI-TOF MS are indicated on the 2-DE map and the PVDF membrane.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Molecular Weight, Infection, Mass Spectrometry

    2D gel electrophoresis and western blot analysis of M. bovis HB0801 whole cell proteins A. Analysis of HB0801 whole cell proteins with 2-DE. Isoelectric points are indicated at the top and molecular weight markers in kDa on the left. B. Immunoblotting patterns of M. bovis HB0801 whole cell proteins obtained using a pool of sera derived from calves experimentally infected with HB0801. The 21 spots identified by MALDI-TOF MS are indicated on the 2D electrophoresis gel and the PVDF membrane.
    Figure Legend Snippet: 2D gel electrophoresis and western blot analysis of M. bovis HB0801 whole cell proteins A. Analysis of HB0801 whole cell proteins with 2-DE. Isoelectric points are indicated at the top and molecular weight markers in kDa on the left. B. Immunoblotting patterns of M. bovis HB0801 whole cell proteins obtained using a pool of sera derived from calves experimentally infected with HB0801. The 21 spots identified by MALDI-TOF MS are indicated on the 2D electrophoresis gel and the PVDF membrane.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Molecular Weight, Derivative Assay, Infection, Mass Spectrometry

    40) Product Images from "A novel de novo MTOR gain-of-function variant in a patient with Smith-Kingsmore syndrome and Antiphospholipid syndrome"

    Article Title: A novel de novo MTOR gain-of-function variant in a patient with Smith-Kingsmore syndrome and Antiphospholipid syndrome

    Journal: European Journal of Human Genetics

    doi: 10.1038/s41431-019-0418-1

    Substrates phosphorylation analysis of mTOR-pathway by western-blot. a Lymphocytes from age-matching control (C), parents (F: father, M: mother), healthy brother (B) and patient (P) were lysed, separated by a gradient 4–20% (w/v) SDS-PAGE, transferred to PVDF membrane (Bio-Rad) and then probed with antibodies against the different proteins. The panels show a representative immunoblotting of mTOR, p-mTOR S2448 or S605 , p-4EBP1 T37/46 , p-S6K S371 , p-S6K T389 , p-ULK1 S758 , p-AKT S473 and β-actin. Densitometry analysis of the peak density normalized over β-actin of p-mTORβ S605 /mTORβ ( b ), p-4EBP1 T37/46 ( c ), p-S6K S371 ( d ), p-S6K T389 ( e ), and p-AKT S473 . Data were the mean (±SD) of three independent experiments, *P
    Figure Legend Snippet: Substrates phosphorylation analysis of mTOR-pathway by western-blot. a Lymphocytes from age-matching control (C), parents (F: father, M: mother), healthy brother (B) and patient (P) were lysed, separated by a gradient 4–20% (w/v) SDS-PAGE, transferred to PVDF membrane (Bio-Rad) and then probed with antibodies against the different proteins. The panels show a representative immunoblotting of mTOR, p-mTOR S2448 or S605 , p-4EBP1 T37/46 , p-S6K S371 , p-S6K T389 , p-ULK1 S758 , p-AKT S473 and β-actin. Densitometry analysis of the peak density normalized over β-actin of p-mTORβ S605 /mTORβ ( b ), p-4EBP1 T37/46 ( c ), p-S6K S371 ( d ), p-S6K T389 ( e ), and p-AKT S473 . Data were the mean (±SD) of three independent experiments, *P

    Techniques Used: Western Blot, SDS Page

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    Electrophoresis:

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    Concentration Assay:

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    p53 T associates with HIF-1. (A) Anti-HIF-1α BN-PAGE immune-blot shows the rate of accumulation of different complexes of HIF-1α at 1% O 2 in HCT116p53+/+ and HCT116p53−/− cells. Purple arrows indicate HIF-1α species (M.W. 120kDa), yellow arrow shows HIF-1 complex (M.W. 212 kDa) and blue arrow suggests p53-HIF-1 complex (M.W. > HIF-1) after an extended run of lysates in 3-15% <t>Bis-tris</t> gradient gel. The black arrow shows higher-order HIF-1α species in HCT116p53+/+ cell line. (B) Foci like structures (yellow arrows) showing co-localization of exogenous HIF-1α (ECFP), HIF-1β (EYFP) and exogenous or endogenous p53 (DsRed Ex or TRITC) in the nucleus of the cell. Scale bar 100μm. (C) Sequestration of endogenous p53 by exogenous HIF-1 subunits in concentration-dependent manner. Scale bar 50μm. Fluorescence images are pseudo-colored and color calibration bars indicate pixel-wise fluorescence intensity. (D) Triple immune reaction-based identification of endogenous p53T-HIF-1 complex. Green arrows indicate complex with M.W. > p53-HIF-1. The black arrow identifies higher order HIF-1α species. Blue, magenta and yellow arrows indicate p53-HIF-1, p53T and HIF-1 complex respectively. Native protein standards were separated from the <t>PVDF</t> membrane post-transfer and stained separately by Coomassie G250. (E) Identification of endogenous p53-HIF-1 complex by cross-reaction of the same immune band against three antibodies by stepwise stripping. anti-p53 DO1 (cyan), anti-HIF-1α (green) and anti-HIF-1β (red) immune blots were merged cautiously in silico to detect cross-reactivity (white). (F) Effect of different detergent combinations on p53 or HIF-1α complexes. Blue arrows indicate p53-HIF-1 complex positions in the immune-blots. Anti-p53 immune-staining confirms dissociation of intact T from p53-HIF-1 complex by D2 detergent (magenta arrow). (G) Schematic representation of the principle of detergent displacement strategy (left panel). Anti-HIF-1α immune blot was stripped for anti-p53 immune detection and two immune blots were cautiously merged in silico to identify the dissociated p53T (magenta) and HIF-1(cyan) entities (dotted yellow circles) (right panel). Higher-order HIF-1α aggregates are shown by black arrows. For the merged anti-p53 immune-blot image, refer to Fig 6D . 3-15% Bis-Tris gradient gel was selected for proper resolution of all complexes in 1D and 2D BN-PAGE run.
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    p53 T associates with HIF-1. (A) Anti-HIF-1α BN-PAGE immune-blot shows the rate of accumulation of different complexes of HIF-1α at 1% O 2 in HCT116p53+/+ and HCT116p53−/− cells. Purple arrows indicate HIF-1α species (M.W. 120kDa), yellow arrow shows HIF-1 complex (M.W. 212 kDa) and blue arrow suggests p53-HIF-1 complex (M.W. > HIF-1) after an extended run of lysates in 3-15% Bis-tris gradient gel. The black arrow shows higher-order HIF-1α species in HCT116p53+/+ cell line. (B) Foci like structures (yellow arrows) showing co-localization of exogenous HIF-1α (ECFP), HIF-1β (EYFP) and exogenous or endogenous p53 (DsRed Ex or TRITC) in the nucleus of the cell. Scale bar 100μm. (C) Sequestration of endogenous p53 by exogenous HIF-1 subunits in concentration-dependent manner. Scale bar 50μm. Fluorescence images are pseudo-colored and color calibration bars indicate pixel-wise fluorescence intensity. (D) Triple immune reaction-based identification of endogenous p53T-HIF-1 complex. Green arrows indicate complex with M.W. > p53-HIF-1. The black arrow identifies higher order HIF-1α species. Blue, magenta and yellow arrows indicate p53-HIF-1, p53T and HIF-1 complex respectively. Native protein standards were separated from the PVDF membrane post-transfer and stained separately by Coomassie G250. (E) Identification of endogenous p53-HIF-1 complex by cross-reaction of the same immune band against three antibodies by stepwise stripping. anti-p53 DO1 (cyan), anti-HIF-1α (green) and anti-HIF-1β (red) immune blots were merged cautiously in silico to detect cross-reactivity (white). (F) Effect of different detergent combinations on p53 or HIF-1α complexes. Blue arrows indicate p53-HIF-1 complex positions in the immune-blots. Anti-p53 immune-staining confirms dissociation of intact T from p53-HIF-1 complex by D2 detergent (magenta arrow). (G) Schematic representation of the principle of detergent displacement strategy (left panel). Anti-HIF-1α immune blot was stripped for anti-p53 immune detection and two immune blots were cautiously merged in silico to identify the dissociated p53T (magenta) and HIF-1(cyan) entities (dotted yellow circles) (right panel). Higher-order HIF-1α aggregates are shown by black arrows. For the merged anti-p53 immune-blot image, refer to Fig 6D . 3-15% Bis-Tris gradient gel was selected for proper resolution of all complexes in 1D and 2D BN-PAGE run.

    Journal: bioRxiv

    Article Title: Oxygen-responsive p53 tetramer-octamer switch controls cell fate

    doi: 10.1101/841668

    Figure Lengend Snippet: p53 T associates with HIF-1. (A) Anti-HIF-1α BN-PAGE immune-blot shows the rate of accumulation of different complexes of HIF-1α at 1% O 2 in HCT116p53+/+ and HCT116p53−/− cells. Purple arrows indicate HIF-1α species (M.W. 120kDa), yellow arrow shows HIF-1 complex (M.W. 212 kDa) and blue arrow suggests p53-HIF-1 complex (M.W. > HIF-1) after an extended run of lysates in 3-15% Bis-tris gradient gel. The black arrow shows higher-order HIF-1α species in HCT116p53+/+ cell line. (B) Foci like structures (yellow arrows) showing co-localization of exogenous HIF-1α (ECFP), HIF-1β (EYFP) and exogenous or endogenous p53 (DsRed Ex or TRITC) in the nucleus of the cell. Scale bar 100μm. (C) Sequestration of endogenous p53 by exogenous HIF-1 subunits in concentration-dependent manner. Scale bar 50μm. Fluorescence images are pseudo-colored and color calibration bars indicate pixel-wise fluorescence intensity. (D) Triple immune reaction-based identification of endogenous p53T-HIF-1 complex. Green arrows indicate complex with M.W. > p53-HIF-1. The black arrow identifies higher order HIF-1α species. Blue, magenta and yellow arrows indicate p53-HIF-1, p53T and HIF-1 complex respectively. Native protein standards were separated from the PVDF membrane post-transfer and stained separately by Coomassie G250. (E) Identification of endogenous p53-HIF-1 complex by cross-reaction of the same immune band against three antibodies by stepwise stripping. anti-p53 DO1 (cyan), anti-HIF-1α (green) and anti-HIF-1β (red) immune blots were merged cautiously in silico to detect cross-reactivity (white). (F) Effect of different detergent combinations on p53 or HIF-1α complexes. Blue arrows indicate p53-HIF-1 complex positions in the immune-blots. Anti-p53 immune-staining confirms dissociation of intact T from p53-HIF-1 complex by D2 detergent (magenta arrow). (G) Schematic representation of the principle of detergent displacement strategy (left panel). Anti-HIF-1α immune blot was stripped for anti-p53 immune detection and two immune blots were cautiously merged in silico to identify the dissociated p53T (magenta) and HIF-1(cyan) entities (dotted yellow circles) (right panel). Higher-order HIF-1α aggregates are shown by black arrows. For the merged anti-p53 immune-blot image, refer to Fig 6D . 3-15% Bis-Tris gradient gel was selected for proper resolution of all complexes in 1D and 2D BN-PAGE run.

    Article Snippet: The proteins were transferred to PVDF membrane (BioRad) in transfer buffer (25mM Tris, 190mM glycine and 0.1% SDS) overnight at 4°C at constant voltage (60V).

    Techniques: Polyacrylamide Gel Electrophoresis, Concentration Assay, Fluorescence, Staining, Stripping Membranes, In Silico

    Metastable p53 T operates via an oxygen-sensitive T⇀O switch. (A) Schematic representation of the CHX trap in a hypoxia gradient. (B, C) To determine metastable p53 T dynamics in response to hypoxia, CHX trap design in (A) was used to capture p53 homo-oligomerization dynamics by anti-p53 BN-PAGE immune blotting at 1, 0.1 or 5% O 2 (immune blot is shown in Fig. 5B ). To sufficiently resolve each homo-oligomer (especially T and O) 5-15% Bis-tris gradient gel (pH 7.0) was utilized. T1 represents duration for which HCT116 p53+/+ cells were exposed to hypoxia before CHX treatment. Purple arrows indicate p53 pool segregated in its constituent homo-oligomers without CHX trap. T2 represents the duration of CHX for hypoxic cells. 24h > T2 > 6h was always maintained for p53T dynamics in 0-72h T1. A red arrow in (B) shows p53 aggregating smears. Native protein standards were run in the same gel and after transfer of samples on PVDF membrane; its lane was cut and stained separately with coomassie brilliant blue G250. Due to inclusion of protein standards in 15 well gel, 60 th h sample for 1% O 2 was analyzed separately or from other replicates. SDS-PAGE based analysis of total p53 pool and GAPDH loading control of immune blots in (B, C) is shown in Fig 5A, B or Fig S3F. (D) R.A. measurements from (B, C) show oxygen-sensitive p53T via shifts in equilibrium state (5% O 2 ). Green and magenta circles correspond to on-off pattern of p53 switch deciphered at 6h. The magenta arrow shows enhanced dimerization or octamerization via T during initial durations that initiates shifts at 1 and 0.1% O 2 respectively. Values and error bars in correspond to mean and standard deviation from three independent replicates of the experiment respectively and are best represented by the immune blots in (B, C) or Fig. 5B .

    Journal: bioRxiv

    Article Title: Oxygen-responsive p53 tetramer-octamer switch controls cell fate

    doi: 10.1101/841668

    Figure Lengend Snippet: Metastable p53 T operates via an oxygen-sensitive T⇀O switch. (A) Schematic representation of the CHX trap in a hypoxia gradient. (B, C) To determine metastable p53 T dynamics in response to hypoxia, CHX trap design in (A) was used to capture p53 homo-oligomerization dynamics by anti-p53 BN-PAGE immune blotting at 1, 0.1 or 5% O 2 (immune blot is shown in Fig. 5B ). To sufficiently resolve each homo-oligomer (especially T and O) 5-15% Bis-tris gradient gel (pH 7.0) was utilized. T1 represents duration for which HCT116 p53+/+ cells were exposed to hypoxia before CHX treatment. Purple arrows indicate p53 pool segregated in its constituent homo-oligomers without CHX trap. T2 represents the duration of CHX for hypoxic cells. 24h > T2 > 6h was always maintained for p53T dynamics in 0-72h T1. A red arrow in (B) shows p53 aggregating smears. Native protein standards were run in the same gel and after transfer of samples on PVDF membrane; its lane was cut and stained separately with coomassie brilliant blue G250. Due to inclusion of protein standards in 15 well gel, 60 th h sample for 1% O 2 was analyzed separately or from other replicates. SDS-PAGE based analysis of total p53 pool and GAPDH loading control of immune blots in (B, C) is shown in Fig 5A, B or Fig S3F. (D) R.A. measurements from (B, C) show oxygen-sensitive p53T via shifts in equilibrium state (5% O 2 ). Green and magenta circles correspond to on-off pattern of p53 switch deciphered at 6h. The magenta arrow shows enhanced dimerization or octamerization via T during initial durations that initiates shifts at 1 and 0.1% O 2 respectively. Values and error bars in correspond to mean and standard deviation from three independent replicates of the experiment respectively and are best represented by the immune blots in (B, C) or Fig. 5B .

    Article Snippet: The proteins were transferred to PVDF membrane (BioRad) in transfer buffer (25mM Tris, 190mM glycine and 0.1% SDS) overnight at 4°C at constant voltage (60V).

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, SDS Page, Standard Deviation

    p53 tetramer exists as the metastable state in basal conditions. (A) Schematic representation of the homo-oligomerization trap generated by CHX (100μM) and MG132. (B) Spontaneous p53 oscillations captured by the trap in the basal state of cells. (C) Anti-p53 BN-PAGE immune blot shows p53 homo-oligomerization in basal state of U2OS cells by −CHX (only MG132 intervention) or +CHX (CHX+MG132 interventions) variants of the trap. 3-17% Bis-tris gradient gel (pH 7.0) shows p53 M, D, T, O and H.O. forms. O is observed as diffused smears. The immune density of O smear shows enhancement with an increase in MG132 dose (μM) in −CHX or +CHX variations. NativeMark protein standards were cut from the PVDF membrane after protein transfer and stained separately with coomassie brilliant blue G250 (CBB) dye. (D) R.A. calculation was performed by the densitometry of immune blots that identifies D↽T (blue arrow) and T⇀O (magenta arrow) conversion as an indicator of metastability of p53 T through −CHX and +CHX trap variants in the basal state of the cells. Immune blot shown in (C) is the best representation of the data in (D). Values and error bars in (D) represent mean and standard deviation from three independent replicates of the experiment respectively.

    Journal: bioRxiv

    Article Title: Oxygen-responsive p53 tetramer-octamer switch controls cell fate

    doi: 10.1101/841668

    Figure Lengend Snippet: p53 tetramer exists as the metastable state in basal conditions. (A) Schematic representation of the homo-oligomerization trap generated by CHX (100μM) and MG132. (B) Spontaneous p53 oscillations captured by the trap in the basal state of cells. (C) Anti-p53 BN-PAGE immune blot shows p53 homo-oligomerization in basal state of U2OS cells by −CHX (only MG132 intervention) or +CHX (CHX+MG132 interventions) variants of the trap. 3-17% Bis-tris gradient gel (pH 7.0) shows p53 M, D, T, O and H.O. forms. O is observed as diffused smears. The immune density of O smear shows enhancement with an increase in MG132 dose (μM) in −CHX or +CHX variations. NativeMark protein standards were cut from the PVDF membrane after protein transfer and stained separately with coomassie brilliant blue G250 (CBB) dye. (D) R.A. calculation was performed by the densitometry of immune blots that identifies D↽T (blue arrow) and T⇀O (magenta arrow) conversion as an indicator of metastability of p53 T through −CHX and +CHX trap variants in the basal state of the cells. Immune blot shown in (C) is the best representation of the data in (D). Values and error bars in (D) represent mean and standard deviation from three independent replicates of the experiment respectively.

    Article Snippet: The proteins were transferred to PVDF membrane (BioRad) in transfer buffer (25mM Tris, 190mM glycine and 0.1% SDS) overnight at 4°C at constant voltage (60V).

    Techniques: Generated, Polyacrylamide Gel Electrophoresis, Staining, Standard Deviation

    Relative Accumulation of MatR and Various Mitochondrial Proteins during Arabidopsis Seed Germination. (A) ). Detection was performed by chemiluminescence with the Image Quant LAS4000 mini analyzer (GE Healthcare). The intensities of protein signals in (A) and (B) ). (B) -PAGE analysis of the respiratory chain complexes during seed germination in Arabidopsis. Crude membrane fractions obtained from dry seeds, imbibed seeds, and mature Arabidopsis seedlings were solubilized with DDM ( n ). For immunodetections, the proteins were transferred from the native gels onto a PVDF membrane (Bio-Rad) in cathode buffer for 15 h at 40 mA, using the Bio-Rad mini transblot cell. The membranes were distained with ethanol before probing with specific antibodies, as indicated below each blot. Arrows indicate the native respiratory complexes, CI (∼1000 kD), CIII (dimer, ∼500 kD), CIV (∼220 kD), and CV (∼600 kD), in Arabidopsis mitochondria. Please note, in (B) , the original COX2 blot has been modified, i.e., the lane corresponding of the mature leaves (M) was cut from the right side of the blot and pasted to the other side (marked with dotted line). No other changes have been made to the original figure.

    Journal: The Plant Cell

    Article Title: The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria

    doi: 10.1105/tpc.16.00398

    Figure Lengend Snippet: Relative Accumulation of MatR and Various Mitochondrial Proteins during Arabidopsis Seed Germination. (A) ). Detection was performed by chemiluminescence with the Image Quant LAS4000 mini analyzer (GE Healthcare). The intensities of protein signals in (A) and (B) ). (B) -PAGE analysis of the respiratory chain complexes during seed germination in Arabidopsis. Crude membrane fractions obtained from dry seeds, imbibed seeds, and mature Arabidopsis seedlings were solubilized with DDM ( n ). For immunodetections, the proteins were transferred from the native gels onto a PVDF membrane (Bio-Rad) in cathode buffer for 15 h at 40 mA, using the Bio-Rad mini transblot cell. The membranes were distained with ethanol before probing with specific antibodies, as indicated below each blot. Arrows indicate the native respiratory complexes, CI (∼1000 kD), CIII (dimer, ∼500 kD), CIV (∼220 kD), and CV (∼600 kD), in Arabidopsis mitochondria. Please note, in (B) , the original COX2 blot has been modified, i.e., the lane corresponding of the mature leaves (M) was cut from the right side of the blot and pasted to the other side (marked with dotted line). No other changes have been made to the original figure.

    Article Snippet: For nondenaturing-PAGE-protein gel blotting, the gel was transferred to a PVDF membrane (Bio-Rad) in Cathode buffer (50 mM Tricine and 15 mM Bis-Tris-HCl, pH 7.0) for 16 h at 4°C (constant current of 40 mA).

    Techniques: Polyacrylamide Gel Electrophoresis, Modification

    RBMY antibody confirmation by using Western-blot analysis of recombinant RBMY. Two concentrations (100 and 10 ng) of each recombinant protein were loaded on SDS-PAGE and after separation were transferred to PVDF membrane. Blots were exposed to X-ray films

    Journal: Iranian Biomedical Journal

    Article Title: Expression Analysis of RNA-Binding Motif Gene on Y Chromosome (RBMY) Protein Isoforms in Testis Tissue and a Testicular Germ Cell Cancer-Derived Cell Line (NT2)

    doi: 10.6091/ibj.1148.2013

    Figure Lengend Snippet: RBMY antibody confirmation by using Western-blot analysis of recombinant RBMY. Two concentrations (100 and 10 ng) of each recombinant protein were loaded on SDS-PAGE and after separation were transferred to PVDF membrane. Blots were exposed to X-ray films

    Article Snippet: Total protein extract (40 µg) from each sample was separated on 12% SDS-polyacrylamide gels for 120 min at 100 V and transferred to PVDF membrane (Bio-Rad, USA) by a wet transfer system (Bio-Rad, USA) at 20 V overnight.

    Techniques: Western Blot, Recombinant, SDS Page

    Compounds 33 and 38 bind Aβ and reduce AβO formation, but have no effect on Aβ production. (A) Representative western blot. Cells were treated were lysed, and proteins were separated by SDS-PAGE. After transfer to a PVDF membrane, blots were probed with the 6E10 antibody. (B) Quantification of total Aβ oligomers from western blotting. Error bars represent SEM. (n=6; ** p

    Journal: Bioorganic & medicinal chemistry

    Article Title: Bivalent ligands incorporating curcumin and diosgenin as multifunctional compounds against Alzheimer’s disease

    doi: 10.1016/j.bmc.2015.10.032

    Figure Lengend Snippet: Compounds 33 and 38 bind Aβ and reduce AβO formation, but have no effect on Aβ production. (A) Representative western blot. Cells were treated were lysed, and proteins were separated by SDS-PAGE. After transfer to a PVDF membrane, blots were probed with the 6E10 antibody. (B) Quantification of total Aβ oligomers from western blotting. Error bars represent SEM. (n=6; ** p

    Article Snippet: Equal amounts of protein (20.0 μg) were separated by SDS-PAGE on a Tris-Tricine gel (Bio-Rad) and transferred onto a PVDF membrane (Bio-Rad).

    Techniques: Western Blot, SDS Page