polyvinylidene difluoride pvdf membrane Search Results


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  • 99
    Thermo Fisher pvdf membranes
    Interaction analysis of trypsin-activated PC with BtR-175 cadherin fragments. ( A ) The induced expression profiles of BtR-175 after Bb and Bt challenge by qRT-PCR. ( B ) Far-western blot analysis of trypsin-activated PC and BtR-175 cadherin fragments. The trypsin-activated PC was separated using 12% (w/v) SDS–PAGE and transferred to <t>PVDF</t> membranes for far-western blot analysis, in which recombinant His-CR7, His-CR11, and His-CR12 proteins (lane 1) were incubated with membranes. The membrane was either incubated with BSA (lane 2) or directly <t>immunoblotted</t> (lane 3) as a negative control. Positive bands were observed only when trypsin-activated PC/CR7 and trypsin-activated PC/CR12 complexes were present. ( C ) His-tag pull-down assays for trypsin-activated PC and BtR-175 cadherin fragments. Lane 1 shows BtR-175 cadherin fragments incubated with PC that were digested with trypsin. The bands indicate the pull-down proteins of PC that bound to PC (CR7 or CR12) complexes. Lane 2 shows an agarose gel incubated with trypsin-activated PC as a control. ( D ) ELISA binding saturation assays of trypsin-activated PC and BtR-175 cadherin fragments. Error bars depict ±SEM. Statistically significant differences from the control samples are indicated; ** P
    Pvdf Membranes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pvdf membranes
    Accumulation of BaMV and satBaMV RNAs and encoded proteins in B. oldhamii protoplasts infected with BaMV alone (V) or co-infected with satBaMV transcripts (F4). (a) Northern blot analysis of BaMV and satBaMV RNAs. At the times indicated, total RNAs were isolated from an equal number of protoplasts, glyoxalated, electrophoresed in a 1 % agarose gel, blotted onto a nylon membrane and probed with 32 P-labelled BaMV-specific ( Lin et al. , 1993 ) or satBaMV-specific ( Lin et al. , 1996 ) probes. The positions of the BaMV genomic RNA (g), subgenomic RNAs (sg1 and sg2) and satBaMV RNA are indicated on the left. rRNA, Total RNA in ethidium bromide-stained gel showing equal loading in each lane. (b) Western blot analysis of BaMV CP and satBaMV P20. From the batch of protoplasts used in (a), total proteins were extracted, resolved by 12.5 % <t>SDS-PAGE</t> and blotted onto <t>PVDF</t> membrane. Blots were probed with rabbit polyclonal anti-BaMV CP or anti-P20 serum followed by incubation with HRP-conjugated anti-rabbit IgG. CP and P20 were detected using an ECL-Plus chemiluminescence system.
    Pvdf Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 57200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad pvdf membrane
    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.
    Pvdf Membrane, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 30744 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore immobilon p pvdf membranes
    Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to <t>PVDF</t> membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.
    Immobilon P Pvdf Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2737 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare pvdf membrane
    Disruption of the rasG gene. ( a ) Schematic representation of the cloning strategy employed to disrupt the rasG gene. A 1.7-kb fragment encoding the cDNA for the blasticidin resistance gene ( bsr ) driven by the constitutive actin15 promoter was inserted by homologous recombination into the rasG promoter between the promoter and the ATG start codon. A probe from the rasG coding sequence ( shaded bar ) was used to detect correct disruptants by Southern blotting of genomic DNA. The expected bands in the parental strain and disruptants are indicated by dotted lines. ( b ) Southern blot of rasG − and wild-type parental genomic DNA. Nuclear DNA from strains IR15 ( rasG − ) and <t>AX2</t> ( wt ) was digested with EcoRI and HindIII, separated on an 0.8% agarose gel, blotted onto nylon, and probed with the rasG coding sequence (see above). The 1.9-kb parental band and 3.2-kb rasG − disrupted band are marked. ( C ) Western blot of rasG − and AX2 wild-type cells. Whole cell lysates were separated by PAGE using a 15% acrylamide gel, blotted onto <t>PVDF,</t> and probed with the general Ras antibody Y13-259 ( left ) and a RasG specific antibody ( right ). Y13-259 recognizes several different Dictyostelium Ras proteins with varying efficiency.
    Pvdf Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 13853 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam pvdf membranes
    Effect of elevated glucose concentrations on the expression of proteins implicated in Zn 2+ homeostasis in mouse pancreatic islets. A , total cell lysates were loaded onto 12% <t>SDS-PAGE,</t> which was subsequently transferred onto a <t>PVDF</t> membrane (see “Experimental Procedures”). The membrane was blotted for ZiP6 (1:200), ZiP7 (1:200), and β-tubulin (1:1000) and with an HRP-lined anti-rabbit (1:5000) secondary antibody. B , quantification of three different immunoblot analyses for ZiP6 and ZiP7. The same area of interest was drawn around the specific bands for ZiP, ZiP7 as well as the corresponding tubulin band, and the intensity was measured using ImageJ software. The ratio of intensity between ZiP or ZiP7 signals and tubulin were plotted. *, p
    Pvdf Membranes, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 5076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology pvdf membranes
    The effect of ageing from 3 months to 12 months on <t>caveolin-1</t> expression in WT mice. Western blot (bottom panel) performed on 30 μg protein from homogenized samples of WT mice indicates no effect of ageing from 3 months to 12 months of caveolin-1 expression. Upper panel indicates actin expression in all samples as assessed by napthol blue black staining of <t>PVDF</t> membrane.
    Pvdf Membranes, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 4930 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc pvdf membrane
    H3.5 exists in human testicular cells within seminiferous tubules. a – c Human testis sections immunohistochemically stained with the anti-H3.5 ( a ), anti-H3.1 ( b ), and anti-H3.3 ( c ) monoclonal antibodies. Bars indicate 50 μm. Arrows and arrow heads in the enlarged picture in panel ( a ) primary spermatocytes at leptotene and preleptotene stages, respectively. d Western blotting. Proteins from isolated sperm were separated by 15 % <t>SDS-PAGE,</t> transferred to a <t>PVDF</t> membrane, and probed with the anti-H3.5 monoclonal antibody ( left panel ) or the anti-H3 C-terminal peptide polyclonal antibody ( right panel ). Recombinant H3.5-H4 and H3.3-H4 complexes were used for controls. Asterisks represent the degraded H3
    Pvdf Membrane, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Merck KGaA pvdf membrane
    Epitope identification through dot-blotting with synthetic peptides. (A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the <t>PVDF</t> membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using <t>RG-M18</t> mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).
    Pvdf Membrane, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 94/100, based on 3182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad immun blot pvdf membrane
    Immunoblot analysis of purified C. difficile toxins A and B. Purified proteins (80 µg each) were subjected to 6% <t>PAGE</t> and transferred onto <t>PVDF</t> membranes. Each membrane was probed using monoclonal primary antibodies specific for toxin A or B. The Pierce ECL Western Blotting Kit was used to detect the bound antibodies. The membrane was exposed to X-ray film (Molecular Technologies, St Louis, MO) and processed using a Konica film processor (Konica Corporation, Tokyo, Japan). Sup, crude culture supernatant; Toxin A, purified toxin A; Toxin B, purified toxin B.
    Immun Blot Pvdf Membrane, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2704 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher polyvinylidene difluoride pvdf membranes
    Deletion of the C-terminal domain increases auto-phosphorylation activity. ( A ) Partial tryptic digestion of recombinant MPK10. 50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at the indicated time points and the reaction was stopped either by adding Laemmli buffer (for N-terminal sequencing) or by lowering the pH to 5.0 and subsequent freezing (for mass determination by SELDI-TOF). For N-terminal sequencing, samples were separated by <t>SDS-PAGE,</t> transferred on <t>PVDF</t> membrane and stained by amidoblack. N-terminal sequencing was performed at the protein analysis platform at the Institut Pasteur. For mass determination, samples were immobilized on a H4 ProteinChip Array (C16 reversed phase surface) and peptide masses identified by SELDI-TOF. Results of the N-terminal sequencing are represented by the cartoon in ( B ), and the sequences are indicated in ( C ). Italic characters represent the Strep3-tag and bold characters represent the sequence of Leishmania major MPK10. White and grey arrowheads indicate respectively lysine or arginine residues recognized by trypsine, including K12, K24, K30 and R392. The white arrow at the position D387 indicates the position of the last cleaved residue resulting in the generation of the form lacking the last 46 amino acids of MPK10. ( D ) In vitro kinase assay using recombinant His-MPK10 (NM) and the truncated kinase mutants His-MPK10-ΔC (ΔC), and His-MPK10-ΔC_K51A (ΔC_K/A). Results are representative of three independent experiments. Purified proteins were incubated with four different substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed at the same time for 30 min at pH 7.5 and 37°C and reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (right), and signals were revealed by auto-radiography with the same exposure time between the different gels (left). The brackets in (D) indicate auto-phosphorylation (Auto-P) and substrate phosphorylation (Substrate-P) signals.
    Polyvinylidene Difluoride Pvdf Membranes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher tropifluor pvdf membrane pore size
    Deletion of the C-terminal domain increases auto-phosphorylation activity. ( A ) Partial tryptic digestion of recombinant MPK10. 50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at the indicated time points and the reaction was stopped either by adding Laemmli buffer (for N-terminal sequencing) or by lowering the pH to 5.0 and subsequent freezing (for mass determination by SELDI-TOF). For N-terminal sequencing, samples were separated by <t>SDS-PAGE,</t> transferred on <t>PVDF</t> membrane and stained by amidoblack. N-terminal sequencing was performed at the protein analysis platform at the Institut Pasteur. For mass determination, samples were immobilized on a H4 ProteinChip Array (C16 reversed phase surface) and peptide masses identified by SELDI-TOF. Results of the N-terminal sequencing are represented by the cartoon in ( B ), and the sequences are indicated in ( C ). Italic characters represent the Strep3-tag and bold characters represent the sequence of Leishmania major MPK10. White and grey arrowheads indicate respectively lysine or arginine residues recognized by trypsine, including K12, K24, K30 and R392. The white arrow at the position D387 indicates the position of the last cleaved residue resulting in the generation of the form lacking the last 46 amino acids of MPK10. ( D ) In vitro kinase assay using recombinant His-MPK10 (NM) and the truncated kinase mutants His-MPK10-ΔC (ΔC), and His-MPK10-ΔC_K51A (ΔC_K/A). Results are representative of three independent experiments. Purified proteins were incubated with four different substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed at the same time for 30 min at pH 7.5 and 37°C and reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (right), and signals were revealed by auto-radiography with the same exposure time between the different gels (left). The brackets in (D) indicate auto-phosphorylation (Auto-P) and substrate phosphorylation (Substrate-P) signals.
    Tropifluor Pvdf Membrane Pore Size, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore immobilon fl pvdf membranes
    Levels of haptoglobin and SAP but not hemopexin, transferrin, or CRP are elevated in the plasma of ASGP-R2(-/-) mice. Equal amounts of plasma protein per sample, 1 μg for hemopexin, 10 μg for haptoglobin, transferrin, and CRP or 100 μg for SAP detection, from 7 Wt and 11 ASGP-R2(-/-) mice were separated by SDS-PAGE and electrophoretically transferred to <t>Immobilon-FL</t> <t>PVDF</t> membranes. Western blots were performed to quantitate the relative amount of: ( panel A ) haptoglobin, ( panel B ) SAP, ( panel C ) hemopexin, ( panel D ) transferrin, and ( panel E ) CRP in each sample. Haptoglobin is elevated 4-fold, p = 0.01, and SAP 2-fold, p = 0.01 in plasma from ASGP-R2(-/-) mice. Hemopexin, transferrin, and CRP did not differ significantly in levels in Wt and ASGP-R2(-/-) mice.
    Immobilon Fl Pvdf Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare hybond p pvdf membrane
    Dot blotting analysis for binding site confirmation. Dot blotting was performed to define the geometric orientations of the selected aptamers. ( a ) LCN2 (74.2 pmol) with 9 selected aptamers (LCN2_Apta1 to LCN2_Apta9, 742 pmol) and controls (C1 to C4) were dotted onto the <t>Hybond-P</t> <t>PVDF</t> membrane. After incubation with the HRP-conjugated anti-LCN2 polyclonal antibody, an assay image was taken using an ECL assay protocol. The plotted signal intensities were calculated using ImageJ software and normalized to C2 (LCN2 74.2 pmol). The schematic epitope binding of two aptamers (LCN2_apta2 and LCN2_apta4) is illustrated in ( b ). All parts of this figure were drawn by the authors K-A. L. and J-Y. A.
    Hybond P Pvdf Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1729 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pall Corporation pvdf membranes
    Purification of AtAPY1 and AtAPY1-δTM. The explanation of the colors in the schematic representations of AtAPY1 and AtAPY1-δTM can be found in Fig. 2 . (A) Total proteins from 1.6 x 10 5 HEK293 cells were harvested at each of the indicated time points post transfection with AtAPY1 DNA, separated in a 4–12% gradient gel under denaturing conditions, transferred onto a <t>PVDF</t> membrane and successively incubated with anti-APY1 and anti-His antibodies (left panel). The black arrows mark the signal specific for AtAPY1, while the gray arrowheads indicate unspecific bands. The right panel shows the total protein extract from 1.4 x 10 8 HEK293 cells harvested at 89 h after transfection with AtAPY1 DNA subjected to Ni 2+ -affinity chromatography. Various fractions were separated in a 4–12% gel under denaturing conditions and either stained with Coomassie or transferred onto a PVDF membrane for Western blot analysis. The black arrow indicates the signal detected with antibodies against AtAPY1. The volumes loaded were 1/480 of the flow through (FT) fraction, 1/50 of each of the final two wash fractions W3 and W4 and 1/100 of the elution fraction E. (B) The left panel shows samples representing equal volumes (1/3,000) of the culture medium of 1 x 10 8 HEK293 cells taken at the indicated time points post transfection with AtAPY1-δTM DNA and separated in a 4–12% gradient gel under denaturing conditions. Subsequently, the proteins were either stained with Coomassie or blotted onto a PVDF membrane for Western blot analysis. The right panel depicts the culture medium of 4 x 10 7 HEK293 cells at time point 88 h after transfection with AtAPY1-δTM DNA subjected to Ni 2+ -affinity chromatography. A gradient gel (4–12%) was loaded with 20 μL of supernatant (S) and 20 μL of flow through (FT), 10 μL of each wash 1–5 and 10 μL of each eluate 1–2. For total volumes of the individual fractions see Materials and Methods . The protein amount loaded for eluate 1 equals about 70 ng. Following <t>SDS-PAGE,</t> the gel was silver-stained.
    Pvdf Membranes, supplied by Pall Corporation, used in various techniques. Bioz Stars score: 93/100, based on 724 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime pvdf membrane
    Purification of AtAPY1 and AtAPY1-δTM. The explanation of the colors in the schematic representations of AtAPY1 and AtAPY1-δTM can be found in Fig. 2 . (A) Total proteins from 1.6 x 10 5 HEK293 cells were harvested at each of the indicated time points post transfection with AtAPY1 DNA, separated in a 4–12% gradient gel under denaturing conditions, transferred onto a <t>PVDF</t> membrane and successively incubated with anti-APY1 and anti-His antibodies (left panel). The black arrows mark the signal specific for AtAPY1, while the gray arrowheads indicate unspecific bands. The right panel shows the total protein extract from 1.4 x 10 8 HEK293 cells harvested at 89 h after transfection with AtAPY1 DNA subjected to Ni 2+ -affinity chromatography. Various fractions were separated in a 4–12% gel under denaturing conditions and either stained with Coomassie or transferred onto a PVDF membrane for Western blot analysis. The black arrow indicates the signal detected with antibodies against AtAPY1. The volumes loaded were 1/480 of the flow through (FT) fraction, 1/50 of each of the final two wash fractions W3 and W4 and 1/100 of the elution fraction E. (B) The left panel shows samples representing equal volumes (1/3,000) of the culture medium of 1 x 10 8 HEK293 cells taken at the indicated time points post transfection with AtAPY1-δTM DNA and separated in a 4–12% gradient gel under denaturing conditions. Subsequently, the proteins were either stained with Coomassie or blotted onto a PVDF membrane for Western blot analysis. The right panel depicts the culture medium of 4 x 10 7 HEK293 cells at time point 88 h after transfection with AtAPY1-δTM DNA subjected to Ni 2+ -affinity chromatography. A gradient gel (4–12%) was loaded with 20 μL of supernatant (S) and 20 μL of flow through (FT), 10 μL of each wash 1–5 and 10 μL of each eluate 1–2. For total volumes of the individual fractions see Materials and Methods . The protein amount loaded for eluate 1 equals about 70 ng. Following <t>SDS-PAGE,</t> the gel was silver-stained.
    Pvdf Membrane, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 753 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Carl Roth GmbH pvdf membranes
    Total membrane fractions of Chara internodal cells. (A) Proteins of membrane fractions (MF) were separated by SDS-PAGE (10%), stained with Coomassie Brilliant Blue (CBB) or blotted onto <t>PVDF</t> membranes for <t>immunodetection</t> of the plasma membrane H + ATPase. 30 μg protein per lane. Numbers on the left refer to molecular weight markers in kDa. Only the upper part of the PVDF membrane was used, the lower part was probed for immunodetection of low molecular weight proteins. (B) Immunodetection of selected organelle marker proteins for vacuoles (VHA-ɛ, H + PPase), ER (BiP2), plasma membrane and endosomal compartments (ARA6) or cytosol (tubulin, GRF 14-3-3). Proteins of the MFs were separated by preparative SDS-PAGE (12.75% or 10% for GRF and ARA6), plotted onto PVDF membranes cut into 3 mm strips and detected with the respective antibodies. 10 μg protein per strip. Molecular weight markers are given in kDa. Arrow heads indicate the expected position of the respective protein.
    Pvdf Membranes, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 93/100, based on 432 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson pvdf membrane
    The Aptamers of F5R1 and F5R2 Enhanced Lysosomal Degradation of α-syn and Rescued the Cell Defects (A) SK-N-SH cells pre-treated with F5R1, F5R2 or random DNA sequence were transfected the α-syn or vector control vectors and incubated for 24 hr. The extracts were separated by <t>SDS-PAGE</t> and blotted onto <t>PVDF</t> membrane. The membrane was blocked and probed with the α-syn specific polyclonal antibody. β-actin served as the loading control. (B) Quantitative analysis of the total protein level of α-syn from (A). (C) SK-N-SH cells were similarly treated as in (A) except for incubation time (48 hr). The cell extracts were immunoblotted with the α-syn polyclonal antibody. β-actin served as the loading control. (D) Quantitative analysis of the total protein level of α-syn from (C). Data are presented as the mean ± SD (one-way ANOVA) ***p
    Pvdf Membrane, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 717 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Interaction analysis of trypsin-activated PC with BtR-175 cadherin fragments. ( A ) The induced expression profiles of BtR-175 after Bb and Bt challenge by qRT-PCR. ( B ) Far-western blot analysis of trypsin-activated PC and BtR-175 cadherin fragments. The trypsin-activated PC was separated using 12% (w/v) SDS–PAGE and transferred to PVDF membranes for far-western blot analysis, in which recombinant His-CR7, His-CR11, and His-CR12 proteins (lane 1) were incubated with membranes. The membrane was either incubated with BSA (lane 2) or directly immunoblotted (lane 3) as a negative control. Positive bands were observed only when trypsin-activated PC/CR7 and trypsin-activated PC/CR12 complexes were present. ( C ) His-tag pull-down assays for trypsin-activated PC and BtR-175 cadherin fragments. Lane 1 shows BtR-175 cadherin fragments incubated with PC that were digested with trypsin. The bands indicate the pull-down proteins of PC that bound to PC (CR7 or CR12) complexes. Lane 2 shows an agarose gel incubated with trypsin-activated PC as a control. ( D ) ELISA binding saturation assays of trypsin-activated PC and BtR-175 cadherin fragments. Error bars depict ±SEM. Statistically significant differences from the control samples are indicated; ** P

    Journal: Scientific Reports

    Article Title: PC, a Novel Oral Insecticidal Toxin from Bacillus bombysepticus Involved in Host Lethality via APN and BtR-175

    doi: 10.1038/srep11101

    Figure Lengend Snippet: Interaction analysis of trypsin-activated PC with BtR-175 cadherin fragments. ( A ) The induced expression profiles of BtR-175 after Bb and Bt challenge by qRT-PCR. ( B ) Far-western blot analysis of trypsin-activated PC and BtR-175 cadherin fragments. The trypsin-activated PC was separated using 12% (w/v) SDS–PAGE and transferred to PVDF membranes for far-western blot analysis, in which recombinant His-CR7, His-CR11, and His-CR12 proteins (lane 1) were incubated with membranes. The membrane was either incubated with BSA (lane 2) or directly immunoblotted (lane 3) as a negative control. Positive bands were observed only when trypsin-activated PC/CR7 and trypsin-activated PC/CR12 complexes were present. ( C ) His-tag pull-down assays for trypsin-activated PC and BtR-175 cadherin fragments. Lane 1 shows BtR-175 cadherin fragments incubated with PC that were digested with trypsin. The bands indicate the pull-down proteins of PC that bound to PC (CR7 or CR12) complexes. Lane 2 shows an agarose gel incubated with trypsin-activated PC as a control. ( D ) ELISA binding saturation assays of trypsin-activated PC and BtR-175 cadherin fragments. Error bars depict ±SEM. Statistically significant differences from the control samples are indicated; ** P

    Article Snippet: After t the proteins were transferred to PVDF membranes, they were immunoblotted with anti-BmAPN4antibody (Invitrogen) or anti-Tubulin mAb (Sigma–Aldrich) following standard procedures.

    Techniques: Expressing, Quantitative RT-PCR, Far Western Blot, SDS Page, Recombinant, Incubation, Negative Control, Agarose Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Binding Assay

    Accumulation of BaMV and satBaMV RNAs and encoded proteins in B. oldhamii protoplasts infected with BaMV alone (V) or co-infected with satBaMV transcripts (F4). (a) Northern blot analysis of BaMV and satBaMV RNAs. At the times indicated, total RNAs were isolated from an equal number of protoplasts, glyoxalated, electrophoresed in a 1 % agarose gel, blotted onto a nylon membrane and probed with 32 P-labelled BaMV-specific ( Lin et al. , 1993 ) or satBaMV-specific ( Lin et al. , 1996 ) probes. The positions of the BaMV genomic RNA (g), subgenomic RNAs (sg1 and sg2) and satBaMV RNA are indicated on the left. rRNA, Total RNA in ethidium bromide-stained gel showing equal loading in each lane. (b) Western blot analysis of BaMV CP and satBaMV P20. From the batch of protoplasts used in (a), total proteins were extracted, resolved by 12.5 % SDS-PAGE and blotted onto PVDF membrane. Blots were probed with rabbit polyclonal anti-BaMV CP or anti-P20 serum followed by incubation with HRP-conjugated anti-rabbit IgG. CP and P20 were detected using an ECL-Plus chemiluminescence system.

    Journal: The Journal of General Virology

    Article Title: Subcellular localization and expression of bamboo mosaic virus satellite RNA-encoded protein

    doi: 10.1099/vir.0.004994-0

    Figure Lengend Snippet: Accumulation of BaMV and satBaMV RNAs and encoded proteins in B. oldhamii protoplasts infected with BaMV alone (V) or co-infected with satBaMV transcripts (F4). (a) Northern blot analysis of BaMV and satBaMV RNAs. At the times indicated, total RNAs were isolated from an equal number of protoplasts, glyoxalated, electrophoresed in a 1 % agarose gel, blotted onto a nylon membrane and probed with 32 P-labelled BaMV-specific ( Lin et al. , 1993 ) or satBaMV-specific ( Lin et al. , 1996 ) probes. The positions of the BaMV genomic RNA (g), subgenomic RNAs (sg1 and sg2) and satBaMV RNA are indicated on the left. rRNA, Total RNA in ethidium bromide-stained gel showing equal loading in each lane. (b) Western blot analysis of BaMV CP and satBaMV P20. From the batch of protoplasts used in (a), total proteins were extracted, resolved by 12.5 % SDS-PAGE and blotted onto PVDF membrane. Blots were probed with rabbit polyclonal anti-BaMV CP or anti-P20 serum followed by incubation with HRP-conjugated anti-rabbit IgG. CP and P20 were detected using an ECL-Plus chemiluminescence system.

    Article Snippet: Total proteins were extracted from transfected protoplasts or infected leaves , separated by 12.5 % SDS-PAGE and electrotransferred to PVDF membranes (Immobilon-P; Millipore).

    Techniques: Infection, Northern Blot, Isolation, Agarose Gel Electrophoresis, Staining, Western Blot, SDS Page, Incubation

    Subcellular localization of HpaM by western blot analysis. Xcc strains were cultured to an OD 600 of 1.0 and proteins were prepared using the method described by Feilmeier and associates (2000) ( A ) or the method described by Chen and associates (2010) ( B ). 30 (for total protein) or 10 μg of protein sample was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The presence of HpaM was detected by anti-His 6 monoclonal antibody. The histidine sensor kinase HpaS and the transcription regulator HpaR1 were used as controls. HpaM, protein sample was prepared from strain ΔhpaM/pR hpaM H6; HpaS, protein sample was prepared from strain ∆hpaS/pR hpaS H6; HpaR1, protein sample was prepared from strain ∆hpaR1/pR hpaR1 H6.

    Journal: Scientific Reports

    Article Title: Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

    doi: 10.1038/srep42724

    Figure Lengend Snippet: Subcellular localization of HpaM by western blot analysis. Xcc strains were cultured to an OD 600 of 1.0 and proteins were prepared using the method described by Feilmeier and associates (2000) ( A ) or the method described by Chen and associates (2010) ( B ). 30 (for total protein) or 10 μg of protein sample was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The presence of HpaM was detected by anti-His 6 monoclonal antibody. The histidine sensor kinase HpaS and the transcription regulator HpaR1 were used as controls. HpaM, protein sample was prepared from strain ΔhpaM/pR hpaM H6; HpaS, protein sample was prepared from strain ∆hpaS/pR hpaS H6; HpaR1, protein sample was prepared from strain ∆hpaR1/pR hpaR1 H6.

    Article Snippet: Briefly, bacterial proteins were separated by 12% (w/v) SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membrane (Millipore Corporation, Billerica, MA, USA).

    Techniques: Western Blot, Cell Culture, SDS Page, Electrophoresis

    HpaM homologues in Xoo and Xoc have similar functions to HpaM. ( A ) HpaM Xoo (HpaM homologue in Xoo ) and HpaM Xoc (HpaM homologue in Xoc ) are essential for type III secretion. Type III secretion signal sequence- gusA fusion reporter plasmid pGUS avrAC was introduced into Xoo and Xoc strains. The resulting recombinant strains were cultured in XOM2 medium for 12 h and the β-glucuronidase (GUS) activities in the culture (Total) and the cultural supernatant (Secreted) were determined. Values are the means ± standard deviation from three repeats. Left and right elements, GUS activities produced by pGUS avrAC in Xoo and Xoc strains, respectively. ( B ) The HpaM homologues HpaM Xoo and HpaM Xoc are also located in the outer membrane. The outer and inner membrane fraction proteins from Xoo and Xoc strains were prepared and 10 μg of each protein sample was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The presence of tested proteins was detected by anti-His 6 monoclonal antibody. The histidine sensor kinase HpaS and transcriptional regulator HpaR1 of Xcc were used as positive and negative controls. OM, outer membrane; IM, inner membrane.

    Journal: Scientific Reports

    Article Title: Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

    doi: 10.1038/srep42724

    Figure Lengend Snippet: HpaM homologues in Xoo and Xoc have similar functions to HpaM. ( A ) HpaM Xoo (HpaM homologue in Xoo ) and HpaM Xoc (HpaM homologue in Xoc ) are essential for type III secretion. Type III secretion signal sequence- gusA fusion reporter plasmid pGUS avrAC was introduced into Xoo and Xoc strains. The resulting recombinant strains were cultured in XOM2 medium for 12 h and the β-glucuronidase (GUS) activities in the culture (Total) and the cultural supernatant (Secreted) were determined. Values are the means ± standard deviation from three repeats. Left and right elements, GUS activities produced by pGUS avrAC in Xoo and Xoc strains, respectively. ( B ) The HpaM homologues HpaM Xoo and HpaM Xoc are also located in the outer membrane. The outer and inner membrane fraction proteins from Xoo and Xoc strains were prepared and 10 μg of each protein sample was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The presence of tested proteins was detected by anti-His 6 monoclonal antibody. The histidine sensor kinase HpaS and transcriptional regulator HpaR1 of Xcc were used as positive and negative controls. OM, outer membrane; IM, inner membrane.

    Article Snippet: Briefly, bacterial proteins were separated by 12% (w/v) SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membrane (Millipore Corporation, Billerica, MA, USA).

    Techniques: Sequencing, Plasmid Preparation, Recombinant, Cell Culture, Standard Deviation, Produced, SDS Page, Electrophoresis

    HpaM is essential for secretion of T3SS effectors in Xcc . Type III secretion signal sequence- gusA fusion reporter plasmids pGUS avrAC and pGUS xopN were introduced into Xcc strains. The resulting recombinant strains were cultured in XVM2 medium for 12 h and the β -glucuronidase (GUS) activities were determined. Values are the means ± standard deviation from three repeats. ( A ) GUS activities in the cultural supernatant (Secreted) and the total culture (Total) produced by pGUS avrAC and pGUS xopN in different background strains. ( B ) Western blot assay. The recombinant plasmid pR avrAC H6, which contains the T3E AvrAC encoding sequence fused with 6×His tag in its C-terminus, was introduced into Xcc strains. The resulting recombinant strains were cultured in XVM2 medium for 12 h and proteins in cultural supernatant (secreted protein) were collected by ultra-filtration using Amicon Ultra-15 centrifugal filter (Millipore Corporation, Billerica, MA, USA) and the total proteins in Xcc cells were prepared as previously described 62 . 30 μg of secreted or cell protein was electrophoresed in SDS-PAGE gel and transferred to a PVDF membrane. The presence of AvrAC was detected by anti-His 6 monoclonal antibody. ( C ) Cya protein translocation assay. The pL avrAC 102 ::CyaA fusion construct was transferred into Xcc strains and the resulting recombinant strains were then used to inoculate Chinese radish ( Raphanus sativus ) leaves. The cAMP level was determined 24 h post-inoculation. Values given are the means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. 8004, wild type strain; ∆hpaM, hpaM deletion mutant; ∆hrcV, hrcV deletion mutant.

    Journal: Scientific Reports

    Article Title: Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

    doi: 10.1038/srep42724

    Figure Lengend Snippet: HpaM is essential for secretion of T3SS effectors in Xcc . Type III secretion signal sequence- gusA fusion reporter plasmids pGUS avrAC and pGUS xopN were introduced into Xcc strains. The resulting recombinant strains were cultured in XVM2 medium for 12 h and the β -glucuronidase (GUS) activities were determined. Values are the means ± standard deviation from three repeats. ( A ) GUS activities in the cultural supernatant (Secreted) and the total culture (Total) produced by pGUS avrAC and pGUS xopN in different background strains. ( B ) Western blot assay. The recombinant plasmid pR avrAC H6, which contains the T3E AvrAC encoding sequence fused with 6×His tag in its C-terminus, was introduced into Xcc strains. The resulting recombinant strains were cultured in XVM2 medium for 12 h and proteins in cultural supernatant (secreted protein) were collected by ultra-filtration using Amicon Ultra-15 centrifugal filter (Millipore Corporation, Billerica, MA, USA) and the total proteins in Xcc cells were prepared as previously described 62 . 30 μg of secreted or cell protein was electrophoresed in SDS-PAGE gel and transferred to a PVDF membrane. The presence of AvrAC was detected by anti-His 6 monoclonal antibody. ( C ) Cya protein translocation assay. The pL avrAC 102 ::CyaA fusion construct was transferred into Xcc strains and the resulting recombinant strains were then used to inoculate Chinese radish ( Raphanus sativus ) leaves. The cAMP level was determined 24 h post-inoculation. Values given are the means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. 8004, wild type strain; ∆hpaM, hpaM deletion mutant; ∆hrcV, hrcV deletion mutant.

    Article Snippet: Briefly, bacterial proteins were separated by 12% (w/v) SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membrane (Millipore Corporation, Billerica, MA, USA).

    Techniques: Sequencing, Recombinant, Cell Culture, Standard Deviation, Produced, Western Blot, Plasmid Preparation, Filtration, SDS Page, Translocation Assay, Construct, Mutagenesis

    Evidence from western blot analysis reveals that HpaM, HrcC and HrcJ are outer and inner membrane-bound proteins, respectively. The outer and inner membrane fraction proteins from strain ∆hrcC/pR hrcC H6 (for HrcC detection), ∆hrcJ/pR hrcJ H6 (for HrcJ detection), and ΔHpaM-HrcC/pR hpaM H6 (for HpaM detection) were prepared. 10 μg of protein for each sample was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The presence of HrcC, HrcJ, and HpaM was detected by anti-His 6 monoclonal antibody. The histidine sensor kinase HpaS (from strain ∆hpaS/pR hpaS H6) was used as a control.

    Journal: Scientific Reports

    Article Title: Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

    doi: 10.1038/srep42724

    Figure Lengend Snippet: Evidence from western blot analysis reveals that HpaM, HrcC and HrcJ are outer and inner membrane-bound proteins, respectively. The outer and inner membrane fraction proteins from strain ∆hrcC/pR hrcC H6 (for HrcC detection), ∆hrcJ/pR hrcJ H6 (for HrcJ detection), and ΔHpaM-HrcC/pR hpaM H6 (for HpaM detection) were prepared. 10 μg of protein for each sample was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The presence of HrcC, HrcJ, and HpaM was detected by anti-His 6 monoclonal antibody. The histidine sensor kinase HpaS (from strain ∆hpaS/pR hpaS H6) was used as a control.

    Article Snippet: Briefly, bacterial proteins were separated by 12% (w/v) SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membrane (Millipore Corporation, Billerica, MA, USA).

    Techniques: Western Blot, SDS Page, Electrophoresis

    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

    Far Western blot analysis of rTsgal binding to IEC proteins. A SDS-PAGE analysis of the IEC proteins. Lane M: The protein molecular weight marker; lane 1: The IEC proteins; lane 2: The C2C12 lysates. B Far Western analysis of the rTsgal binding to IEC proteins. The IEC proteins blotted on the PVDF membrane was pre-incubated with rTsgal (lane 1–3), PBS (lane 4–6), C2C12 proteins was also pre-incubated with rTsgal (lane 7–9). Subsequently, both cell proteins were incubated with anti-rTsgal serum (lane 1, 4 and 7), infection serum (lane 2, 5 and 8) or normal serum (lane 3, 6 and 9), respectively. The binding between rTsgal and IECs was detected only with anti-rTsgal serum (lane 1) and infection serum (lane 2).

    Journal: Veterinary Research

    Article Title: Molecular characterization of Trichinella spiralis galectin and its participation in larval invasion of host’s intestinal epithelial cells

    doi: 10.1186/s13567-018-0573-3

    Figure Lengend Snippet: Far Western blot analysis of rTsgal binding to IEC proteins. A SDS-PAGE analysis of the IEC proteins. Lane M: The protein molecular weight marker; lane 1: The IEC proteins; lane 2: The C2C12 lysates. B Far Western analysis of the rTsgal binding to IEC proteins. The IEC proteins blotted on the PVDF membrane was pre-incubated with rTsgal (lane 1–3), PBS (lane 4–6), C2C12 proteins was also pre-incubated with rTsgal (lane 7–9). Subsequently, both cell proteins were incubated with anti-rTsgal serum (lane 1, 4 and 7), infection serum (lane 2, 5 and 8) or normal serum (lane 3, 6 and 9), respectively. The binding between rTsgal and IECs was detected only with anti-rTsgal serum (lane 1) and infection serum (lane 2).

    Article Snippet: Western blot analysis Samples containing somatic proteins of ML, IIL, AW and NBL, ML ES proteins and rTsgal protein were separated by SDS-PAGE with a 12% separating gel and then were transferred onto the polyvinylidene difluoride (PVDF) membranes using a Mini Trans-Blot® Cell (Bio-Rad, China) at 250 mA for 1.5 h [ ].

    Techniques: Far Western Blot, Binding Assay, SDS Page, Molecular Weight, Marker, Western Blot, Incubation, Infection

    Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae *

    doi: 10.1074/jbc.M114.633131

    Figure Lengend Snippet: Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.

    Article Snippet: Protein samples incubated with SDS sample buffer (50 m m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 1.33% β-mercaptoethanol) were separated on 12% Tris-glycine SDS-polyacrylamide gels by electrophoresis ( ) followed by transfer to Immobilon-P PVDF membranes (Millipore) at 100 V for 1.5 h. Membranes were then blocked overnight in 3% nonfat milk, phosphate-buffered saline (140.7 m m NaCl, 9.3 m m Na2 HPO4 , pH 7.4), 0.1% Tween 20.

    Techniques: Purification, Affinity Purification, SDS Page

    CNAP-tagged Coq proteins co-precipitate several other Coq proteins. W303, CNAP3, CNAP6, and CNAP9 purified mitochondria (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification with Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting with antisera to the designated yeast polypeptides. 25 μg of mitochondria protein were analyzed for each strain ( M ), and 2.5% of the first anti-PC elution ( E1 ) volume was loaded per strain (25 μl). Arrows denote each tagged protein in their respective blots. The predominant band in the Coq3 blot detected in the mitochondrial samples represents a background protein and not Coq3, accounting for its presence in CNAP3 mitochondria.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae *

    doi: 10.1074/jbc.M114.633131

    Figure Lengend Snippet: CNAP-tagged Coq proteins co-precipitate several other Coq proteins. W303, CNAP3, CNAP6, and CNAP9 purified mitochondria (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification with Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting with antisera to the designated yeast polypeptides. 25 μg of mitochondria protein were analyzed for each strain ( M ), and 2.5% of the first anti-PC elution ( E1 ) volume was loaded per strain (25 μl). Arrows denote each tagged protein in their respective blots. The predominant band in the Coq3 blot detected in the mitochondrial samples represents a background protein and not Coq3, accounting for its presence in CNAP3 mitochondria.

    Article Snippet: Protein samples incubated with SDS sample buffer (50 m m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 1.33% β-mercaptoethanol) were separated on 12% Tris-glycine SDS-polyacrylamide gels by electrophoresis ( ) followed by transfer to Immobilon-P PVDF membranes (Millipore) at 100 V for 1.5 h. Membranes were then blocked overnight in 3% nonfat milk, phosphate-buffered saline (140.7 m m NaCl, 9.3 m m Na2 HPO4 , pH 7.4), 0.1% Tween 20.

    Techniques: Purification, Affinity Purification, SDS Page

    Antibodies to different Sec6 subdomains label distinct subcellular structures. (A) Immunofluorescent labeling of MDCK cells with antibodies to Sec6 NT1 (mAb 13F10), NT2 (mAb 16G4), CT1 (mAb 10C3), CT2a (mAb 3F3), CT2b (mAb 11A2), or CT3 (mAb 8A5) subdomains (“α-Sec6”) or hybridoma supernatants that were pre-incubated with recombinant Sec6 fragments containing epitopes recognized by those antibodies (“depleted”). Additional mAbs to Sec6 NT1 (mAb 8F9), NT2 (mAb 22F5), and CT1 (mAb 4C8) subdomains produced immunolocalization patterns that were indistinguishable from those of other mAbs that bound within the same subdomain. Cells were fixed with either 4% paraformaldehyde (NT1 and CT1) or 100% methanol (NT2, CT2a, CT2b, and CT3). For NT1 and NT2 labeling, cells were extracted with 1% Triton X-100 prior to fixation, and in top and bottom panels DNA was labeled with DAPI (blue). Bars = 10 μm. (B) Immunoblotting of Sec6 in detergent lysates of MDCK cells. Proteins (3, 1, 0.3, or 0.1 μg) were separated by SDS-PAGE and electrophoretically transferred to Immobilon P membranes. Membranes were probed with pooled antibodies to indicated Sec6 subdomains (“α-Sec6”), or with hybridoma supernatants that were pre-incubated with recombinant Sec6 fragments containing epitopes recognized by those antibodies (“Depleted”). An immunoblot probed for β-tubulin is shown as a loading control. Protein standards indicated are β-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), and carbonic anhydrase (29 kDa).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies

    doi: 10.3389/fcell.2016.00051

    Figure Lengend Snippet: Antibodies to different Sec6 subdomains label distinct subcellular structures. (A) Immunofluorescent labeling of MDCK cells with antibodies to Sec6 NT1 (mAb 13F10), NT2 (mAb 16G4), CT1 (mAb 10C3), CT2a (mAb 3F3), CT2b (mAb 11A2), or CT3 (mAb 8A5) subdomains (“α-Sec6”) or hybridoma supernatants that were pre-incubated with recombinant Sec6 fragments containing epitopes recognized by those antibodies (“depleted”). Additional mAbs to Sec6 NT1 (mAb 8F9), NT2 (mAb 22F5), and CT1 (mAb 4C8) subdomains produced immunolocalization patterns that were indistinguishable from those of other mAbs that bound within the same subdomain. Cells were fixed with either 4% paraformaldehyde (NT1 and CT1) or 100% methanol (NT2, CT2a, CT2b, and CT3). For NT1 and NT2 labeling, cells were extracted with 1% Triton X-100 prior to fixation, and in top and bottom panels DNA was labeled with DAPI (blue). Bars = 10 μm. (B) Immunoblotting of Sec6 in detergent lysates of MDCK cells. Proteins (3, 1, 0.3, or 0.1 μg) were separated by SDS-PAGE and electrophoretically transferred to Immobilon P membranes. Membranes were probed with pooled antibodies to indicated Sec6 subdomains (“α-Sec6”), or with hybridoma supernatants that were pre-incubated with recombinant Sec6 fragments containing epitopes recognized by those antibodies (“Depleted”). An immunoblot probed for β-tubulin is shown as a loading control. Protein standards indicated are β-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), and carbonic anhydrase (29 kDa).

    Article Snippet: Proteins were electrophoretically transferred onto Immobilon PVDF membrane (Millipore) and blocked with Blotto (5% nonfat dry milk, 0.5% normal goat serum, and 0.1% sodium azide in TBS) overnight at 4°C.

    Techniques: Labeling, Incubation, Recombinant, Produced, SDS Page

    Sec6 is quantitatively associated with Sec8 in high molecular weight complexes in MDCK cells . (A) All endogenous Sec6 is bound to Sec8. MDCK RIPA extracts were immunoprecipitated 4 times with anti-Sec8-bound protein A sepharose. Input lysate represents 10% of starting material. Depleted lysate (“spent”) represents 50% of the final post-immunoprecipitation supernatant. Samples were resolved by SDS-PAGE and immunoblotted with anti-Sec8(8F12) and anti-Sec6(10D11) antibodies. (B) Detergent extracts of polarized MDCK cells were fractionated by Superose 6 FPLC as described in Experimental Procedures. Fractions 9–27 were divided into equal aliquots, separated by SDS-PAGE, and transferred to Immobilon P membranes. Membranes were probed with anti-Sec8(8F12) and anti-Sec6(10D11) antibodies. Elution peaks of globular protein standards with known molecular weights were also determined: thyroglobulin, Mr = 669,000 (fraction 16); apoferritin, Mr = 443,000 (fraction 19); catalase, Mr = 232,000 (fraction 22); bovine serum albumin, Mr = 66,000 (fraction 25).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies

    doi: 10.3389/fcell.2016.00051

    Figure Lengend Snippet: Sec6 is quantitatively associated with Sec8 in high molecular weight complexes in MDCK cells . (A) All endogenous Sec6 is bound to Sec8. MDCK RIPA extracts were immunoprecipitated 4 times with anti-Sec8-bound protein A sepharose. Input lysate represents 10% of starting material. Depleted lysate (“spent”) represents 50% of the final post-immunoprecipitation supernatant. Samples were resolved by SDS-PAGE and immunoblotted with anti-Sec8(8F12) and anti-Sec6(10D11) antibodies. (B) Detergent extracts of polarized MDCK cells were fractionated by Superose 6 FPLC as described in Experimental Procedures. Fractions 9–27 were divided into equal aliquots, separated by SDS-PAGE, and transferred to Immobilon P membranes. Membranes were probed with anti-Sec8(8F12) and anti-Sec6(10D11) antibodies. Elution peaks of globular protein standards with known molecular weights were also determined: thyroglobulin, Mr = 669,000 (fraction 16); apoferritin, Mr = 443,000 (fraction 19); catalase, Mr = 232,000 (fraction 22); bovine serum albumin, Mr = 66,000 (fraction 25).

    Article Snippet: Proteins were electrophoretically transferred onto Immobilon PVDF membrane (Millipore) and blocked with Blotto (5% nonfat dry milk, 0.5% normal goat serum, and 0.1% sodium azide in TBS) overnight at 4°C.

    Techniques: Molecular Weight, Immunoprecipitation, SDS Page, Fast Protein Liquid Chromatography

    Effect of E3-6.7K on the induction of procaspase-3 processing and PARP cleavage during TNF-induced apoptosis in vivo. Cell extracts were obtained from U937-neo and U937-6.7K cells that had been treated with 10 ng of TNF and 0.5 μg of CHX per ml for various lengths of time. Lysates containing equivalent amounts of protein based on the bicinchoninic acid (Pierce) protein concentration assay were loaded in each lane. After electrophoresis and transfer to PVDF membranes, blots were incubated with anti-caspase-3 rabbit antiserum that recognizes the 17- and 11-kDa subunits of the active, processed protein (A) and anti-PARP mouse monoclonal antibody that recognizes both the active 116-kDa and inactive 85-kDa forms of the protein (B). The blots were developed with a secondary antibody and visualized by chemiluminescence (Pierce Chemical). Similar results were obtained in two repeat experiments.

    Journal: Journal of Virology

    Article Title: Adenovirus E3-6.7K Maintains Calcium Homeostasis and Prevents Apoptosis and Arachidonic Acid Release

    doi: 10.1128/JVI.76.4.1578-1587.2002

    Figure Lengend Snippet: Effect of E3-6.7K on the induction of procaspase-3 processing and PARP cleavage during TNF-induced apoptosis in vivo. Cell extracts were obtained from U937-neo and U937-6.7K cells that had been treated with 10 ng of TNF and 0.5 μg of CHX per ml for various lengths of time. Lysates containing equivalent amounts of protein based on the bicinchoninic acid (Pierce) protein concentration assay were loaded in each lane. After electrophoresis and transfer to PVDF membranes, blots were incubated with anti-caspase-3 rabbit antiserum that recognizes the 17- and 11-kDa subunits of the active, processed protein (A) and anti-PARP mouse monoclonal antibody that recognizes both the active 116-kDa and inactive 85-kDa forms of the protein (B). The blots were developed with a secondary antibody and visualized by chemiluminescence (Pierce Chemical). Similar results were obtained in two repeat experiments.

    Article Snippet: Equivalent amounts of protein from each sample were resolved on a 10% glycine-SDS-polyacrylamide gel electrophoresis (PAGE) Laemmli gel system, blotted with the Towbin vertical-transfer wet system onto a 0.45-μm-pore-size Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore), and incubated with anti-PARP mouse monoclonal antibody (1:5,000 dilution; Pharmingen).

    Techniques: In Vivo, Protein Concentration, Electrophoresis, Incubation

    Disruption of the rasG gene. ( a ) Schematic representation of the cloning strategy employed to disrupt the rasG gene. A 1.7-kb fragment encoding the cDNA for the blasticidin resistance gene ( bsr ) driven by the constitutive actin15 promoter was inserted by homologous recombination into the rasG promoter between the promoter and the ATG start codon. A probe from the rasG coding sequence ( shaded bar ) was used to detect correct disruptants by Southern blotting of genomic DNA. The expected bands in the parental strain and disruptants are indicated by dotted lines. ( b ) Southern blot of rasG − and wild-type parental genomic DNA. Nuclear DNA from strains IR15 ( rasG − ) and AX2 ( wt ) was digested with EcoRI and HindIII, separated on an 0.8% agarose gel, blotted onto nylon, and probed with the rasG coding sequence (see above). The 1.9-kb parental band and 3.2-kb rasG − disrupted band are marked. ( C ) Western blot of rasG − and AX2 wild-type cells. Whole cell lysates were separated by PAGE using a 15% acrylamide gel, blotted onto PVDF, and probed with the general Ras antibody Y13-259 ( left ) and a RasG specific antibody ( right ). Y13-259 recognizes several different Dictyostelium Ras proteins with varying efficiency.

    Journal: The Journal of Cell Biology

    Article Title: Dictyostelium RasG Is Required for Normal Motility and Cytokinesis, But Not Growth

    doi:

    Figure Lengend Snippet: Disruption of the rasG gene. ( a ) Schematic representation of the cloning strategy employed to disrupt the rasG gene. A 1.7-kb fragment encoding the cDNA for the blasticidin resistance gene ( bsr ) driven by the constitutive actin15 promoter was inserted by homologous recombination into the rasG promoter between the promoter and the ATG start codon. A probe from the rasG coding sequence ( shaded bar ) was used to detect correct disruptants by Southern blotting of genomic DNA. The expected bands in the parental strain and disruptants are indicated by dotted lines. ( b ) Southern blot of rasG − and wild-type parental genomic DNA. Nuclear DNA from strains IR15 ( rasG − ) and AX2 ( wt ) was digested with EcoRI and HindIII, separated on an 0.8% agarose gel, blotted onto nylon, and probed with the rasG coding sequence (see above). The 1.9-kb parental band and 3.2-kb rasG − disrupted band are marked. ( C ) Western blot of rasG − and AX2 wild-type cells. Whole cell lysates were separated by PAGE using a 15% acrylamide gel, blotted onto PVDF, and probed with the general Ras antibody Y13-259 ( left ) and a RasG specific antibody ( right ). Y13-259 recognizes several different Dictyostelium Ras proteins with varying efficiency.

    Article Snippet: Western Blotting rasG − + AX2 cell lysates were separated by SDS-PAGE and blotted onto 0.45 μm PVDF membrane ( Amersham Life Science, Pittsburgh, PA) by standard procedures ( ).

    Techniques: Clone Assay, Homologous Recombination, Sequencing, Southern Blot, Agarose Gel Electrophoresis, Western Blot, Polyacrylamide Gel Electrophoresis, Acrylamide Gel Assay

    Western blotting confirms the presence of ApoE and Na + /K + -ATPase α-chains in SAF preparations. Based on an estimate of 10 µg PrP Sc per brain, the equivalent of 0.25 µg/µl PrP Sc from an ME7 (lane 1), 22F (lane 2) and 79A (lane 3) SAF preparation were resolved by SDS-PAGE, blotted onto PVDF membrane and probed with the primary antibody against (A) Apolipoprotein E (B) Total Na + /K + ATPase α-chains using a pan α-chain antibody (C) Na + /K + ATPase α2 isoform and (D) Na + /K + ATPase α3 isoform. In all cases, in lanes 4 and 5 were loaded the equivalent of 0.25 µg/µl of control preparation material from uninfected WT and PrP −/− mouse brains respectively. Molecular weight markers are in kDa.

    Journal: PLoS ONE

    Article Title: Na+/K+-ATPase Is Present in Scrapie-Associated Fibrils, Modulates PrP Misfolding In Vitro and Links PrP Function and Dysfunction

    doi: 10.1371/journal.pone.0026813

    Figure Lengend Snippet: Western blotting confirms the presence of ApoE and Na + /K + -ATPase α-chains in SAF preparations. Based on an estimate of 10 µg PrP Sc per brain, the equivalent of 0.25 µg/µl PrP Sc from an ME7 (lane 1), 22F (lane 2) and 79A (lane 3) SAF preparation were resolved by SDS-PAGE, blotted onto PVDF membrane and probed with the primary antibody against (A) Apolipoprotein E (B) Total Na + /K + ATPase α-chains using a pan α-chain antibody (C) Na + /K + ATPase α2 isoform and (D) Na + /K + ATPase α3 isoform. In all cases, in lanes 4 and 5 were loaded the equivalent of 0.25 µg/µl of control preparation material from uninfected WT and PrP −/− mouse brains respectively. Molecular weight markers are in kDa.

    Article Snippet: For Western blotting, gels were semi-dry blotted onto Immobilon-P PVDF membrane (Whatman).

    Techniques: Western Blot, SDS Page, Molecular Weight

    Effect of elevated glucose concentrations on the expression of proteins implicated in Zn 2+ homeostasis in mouse pancreatic islets. A , total cell lysates were loaded onto 12% SDS-PAGE, which was subsequently transferred onto a PVDF membrane (see “Experimental Procedures”). The membrane was blotted for ZiP6 (1:200), ZiP7 (1:200), and β-tubulin (1:1000) and with an HRP-lined anti-rabbit (1:5000) secondary antibody. B , quantification of three different immunoblot analyses for ZiP6 and ZiP7. The same area of interest was drawn around the specific bands for ZiP, ZiP7 as well as the corresponding tubulin band, and the intensity was measured using ImageJ software. The ratio of intensity between ZiP or ZiP7 signals and tubulin were plotted. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Glucose Regulates Free Cytosolic Zn2+ Concentration, Slc39 (ZiP), and Metallothionein Gene Expression in Primary Pancreatic Islet ?-Cells *

    doi: 10.1074/jbc.M111.246082

    Figure Lengend Snippet: Effect of elevated glucose concentrations on the expression of proteins implicated in Zn 2+ homeostasis in mouse pancreatic islets. A , total cell lysates were loaded onto 12% SDS-PAGE, which was subsequently transferred onto a PVDF membrane (see “Experimental Procedures”). The membrane was blotted for ZiP6 (1:200), ZiP7 (1:200), and β-tubulin (1:1000) and with an HRP-lined anti-rabbit (1:5000) secondary antibody. B , quantification of three different immunoblot analyses for ZiP6 and ZiP7. The same area of interest was drawn around the specific bands for ZiP, ZiP7 as well as the corresponding tubulin band, and the intensity was measured using ImageJ software. The ratio of intensity between ZiP or ZiP7 signals and tubulin were plotted. *, p

    Article Snippet: Total protein extracts (30 μg) were resolved by SDS-PAGE (12% v/v acrylamide) and transferred to PVDF membranes, followed by immunoblotting with rabbit polyclonal anti mLIV-1 (ZiP6) and anti mKE4 (ZiP7, both used 1:200, Ref. ), mouse monoclonal anti-metallothioneins (1:500, Abcam), and mouse monoclonal anti-tubulin (1:5000, Sigma clone B-5-1-2) antibodies.

    Techniques: Expressing, SDS Page, Software

    The effect of ageing from 3 months to 12 months on caveolin-1 expression in WT mice. Western blot (bottom panel) performed on 30 μg protein from homogenized samples of WT mice indicates no effect of ageing from 3 months to 12 months of caveolin-1 expression. Upper panel indicates actin expression in all samples as assessed by napthol blue black staining of PVDF membrane.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Age-related changes in the contractile and passive arterial properties of murine mesenteric small arteries are altered by caveolin-1 knockout

    doi: 10.1111/j.1582-4934.2011.01457.x

    Figure Lengend Snippet: The effect of ageing from 3 months to 12 months on caveolin-1 expression in WT mice. Western blot (bottom panel) performed on 30 μg protein from homogenized samples of WT mice indicates no effect of ageing from 3 months to 12 months of caveolin-1 expression. Upper panel indicates actin expression in all samples as assessed by napthol blue black staining of PVDF membrane.

    Article Snippet: SDS-PAGE/Western blotting (10% acrylamide gels) was performed and PVDF membranes probed with rabbit polyclonal anti-caveolin-1 primary antibody (1:1000, sc894, Lot number B0409; Santa Cruz, Heidelberg, Germany) and goat anti-rabbit horseradish-peroxidase conjugated secondary antibody (1:2000, product number P0448; Dako UK Ltd., Camdridgeshire, UK).

    Techniques: Expressing, Mouse Assay, Western Blot, Staining

    H3.5 exists in human testicular cells within seminiferous tubules. a – c Human testis sections immunohistochemically stained with the anti-H3.5 ( a ), anti-H3.1 ( b ), and anti-H3.3 ( c ) monoclonal antibodies. Bars indicate 50 μm. Arrows and arrow heads in the enlarged picture in panel ( a ) primary spermatocytes at leptotene and preleptotene stages, respectively. d Western blotting. Proteins from isolated sperm were separated by 15 % SDS-PAGE, transferred to a PVDF membrane, and probed with the anti-H3.5 monoclonal antibody ( left panel ) or the anti-H3 C-terminal peptide polyclonal antibody ( right panel ). Recombinant H3.5-H4 and H3.3-H4 complexes were used for controls. Asterisks represent the degraded H3

    Journal: Epigenetics & Chromatin

    Article Title: Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis

    doi: 10.1186/s13072-016-0051-y

    Figure Lengend Snippet: H3.5 exists in human testicular cells within seminiferous tubules. a – c Human testis sections immunohistochemically stained with the anti-H3.5 ( a ), anti-H3.1 ( b ), and anti-H3.3 ( c ) monoclonal antibodies. Bars indicate 50 μm. Arrows and arrow heads in the enlarged picture in panel ( a ) primary spermatocytes at leptotene and preleptotene stages, respectively. d Western blotting. Proteins from isolated sperm were separated by 15 % SDS-PAGE, transferred to a PVDF membrane, and probed with the anti-H3.5 monoclonal antibody ( left panel ) or the anti-H3 C-terminal peptide polyclonal antibody ( right panel ). Recombinant H3.5-H4 and H3.3-H4 complexes were used for controls. Asterisks represent the degraded H3

    Article Snippet: The proteins in the cell lysate were fractionated by 15 % SDS-PAGE, transferred to a PVDF membrane, and detected with an anti-H3 rabbit polyclonal antibody (#9715, Cell Signaling Technology, Inc.) or the culture supernatant of the hybridoma producing the anti-H3.5 monoclonal antibody (1:100 dilution).

    Techniques: Staining, Western Blot, Isolation, SDS Page, Recombinant

    Epitope identification through dot-blotting with synthetic peptides. (A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the PVDF membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using RG-M18 mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).

    Journal: PLoS ONE

    Article Title: Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection

    doi: 10.1371/journal.pone.0126121

    Figure Lengend Snippet: Epitope identification through dot-blotting with synthetic peptides. (A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the PVDF membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using RG-M18 mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).

    Article Snippet: After blocking with 5% skimmed milk in TBST buffer at 37°C for 30 min, the PVDF membrane was incubated with 1:1,000 diluted RG-M18 mAB in 5% skimmed milk at 37°C for 1 h. The membrane was then washed, and subjected to secondary antibody hybridization with polyclonal goat anti-mouse immunoglobulin/AP at a dilution of 1:5,000 in TBST buffer for 1 h at 37°C.

    Techniques: Peptide Microarray, Positive Control

    Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.

    Article Snippet: SDS–PAGE and Western blotting Samples were separated by SDS–PAGE on 4-12% Bis-Tris polyacrylamide gels (Life Technologies) using PAGE Ruler (Thermo Fisher Scientific) as a size marker and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA).

    Techniques: Cell Culture, SDS Page

    Calpain cleaves otoferlin and myoferlin in addition to dysferlin. (A) Calpain rapidly cleaves immunoprecipitated ferlin proteins in vitro. Dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag were immunoprecipitated with anti-myc and protein G–Sepharose (see Materials and Methods ). Dysferlin-bound Sepharose beads were incubated with purified 0.2 A.U. of recombinant calpain-1 at 30°C for 2 or 10 s in the presence of 2 mM CaCl 2. Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94°C. Digested samples were analyzed by SDS–PAGE and Western blot. Top, C-terminal fragments detected with anti-myc (dysferlin) or anti-Flag (myoferlin and otoferlin). Bottom, N-terminal fragments detected by N-terminal (Romeo-dysferlin) or internal antibodies (7D2, myoferlin; C12, otoferlin). (B) Dysferlin and otoferlin display damage-dependent cleavage, whereas myoferlin cleavage appears to be constitutive. HEK293 cells were transfected with dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag and lysed in calcium-free RIPA (lane 1), RIPA containing 900 μM calcium (permissive for calpain cleavage), or damaged by scraping in the presence of calcium. Scraped cell pellets were lysed in calcium-free RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. Dysferlin was detected with anti-Myc; otoferlin and myoferlin were detected with anti-Flag. (C) Diagram of the predicted calpain cleavage sites within dysferlin, otoferlin, and myoferlin (schematic produced using DOG 2.0; Ren et al. , 2009 ). Molecular weight calculation of the cleaved C-terminal modules was used to elucidate the most likely calpain cleavage site ( ccd.biocuckoo.org ). In each case, the C-terminal fragments released by calpain cleavage represent transmembrane-anchored, dual-C2-domain modules.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Calpain cleaves otoferlin and myoferlin in addition to dysferlin. (A) Calpain rapidly cleaves immunoprecipitated ferlin proteins in vitro. Dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag were immunoprecipitated with anti-myc and protein G–Sepharose (see Materials and Methods ). Dysferlin-bound Sepharose beads were incubated with purified 0.2 A.U. of recombinant calpain-1 at 30°C for 2 or 10 s in the presence of 2 mM CaCl 2. Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94°C. Digested samples were analyzed by SDS–PAGE and Western blot. Top, C-terminal fragments detected with anti-myc (dysferlin) or anti-Flag (myoferlin and otoferlin). Bottom, N-terminal fragments detected by N-terminal (Romeo-dysferlin) or internal antibodies (7D2, myoferlin; C12, otoferlin). (B) Dysferlin and otoferlin display damage-dependent cleavage, whereas myoferlin cleavage appears to be constitutive. HEK293 cells were transfected with dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag and lysed in calcium-free RIPA (lane 1), RIPA containing 900 μM calcium (permissive for calpain cleavage), or damaged by scraping in the presence of calcium. Scraped cell pellets were lysed in calcium-free RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. Dysferlin was detected with anti-Myc; otoferlin and myoferlin were detected with anti-Flag. (C) Diagram of the predicted calpain cleavage sites within dysferlin, otoferlin, and myoferlin (schematic produced using DOG 2.0; Ren et al. , 2009 ). Molecular weight calculation of the cleaved C-terminal modules was used to elucidate the most likely calpain cleavage site ( ccd.biocuckoo.org ). In each case, the C-terminal fragments released by calpain cleavage represent transmembrane-anchored, dual-C2-domain modules.

    Article Snippet: SDS–PAGE and Western blotting Samples were separated by SDS–PAGE on 4-12% Bis-Tris polyacrylamide gels (Life Technologies) using PAGE Ruler (Thermo Fisher Scientific) as a size marker and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA).

    Techniques: Immunoprecipitation, In Vitro, Incubation, Purification, Recombinant, Lysis, SDS Page, Western Blot, Transfection, Produced, Molecular Weight

    Immunoblot analysis of purified C. difficile toxins A and B. Purified proteins (80 µg each) were subjected to 6% PAGE and transferred onto PVDF membranes. Each membrane was probed using monoclonal primary antibodies specific for toxin A or B. The Pierce ECL Western Blotting Kit was used to detect the bound antibodies. The membrane was exposed to X-ray film (Molecular Technologies, St Louis, MO) and processed using a Konica film processor (Konica Corporation, Tokyo, Japan). Sup, crude culture supernatant; Toxin A, purified toxin A; Toxin B, purified toxin B.

    Journal: PLoS ONE

    Article Title: Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins

    doi: 10.1371/journal.pone.0079631

    Figure Lengend Snippet: Immunoblot analysis of purified C. difficile toxins A and B. Purified proteins (80 µg each) were subjected to 6% PAGE and transferred onto PVDF membranes. Each membrane was probed using monoclonal primary antibodies specific for toxin A or B. The Pierce ECL Western Blotting Kit was used to detect the bound antibodies. The membrane was exposed to X-ray film (Molecular Technologies, St Louis, MO) and processed using a Konica film processor (Konica Corporation, Tokyo, Japan). Sup, crude culture supernatant; Toxin A, purified toxin A; Toxin B, purified toxin B.

    Article Snippet: Purified C. difficile toxins A and B (80 µg each) were separated on 6% polyacrylamide electrophoresis (PAGE) gels and transferred onto Immun-Blot PVDF membrane (BioRad, Hercules, CA) using a Trans-Blot cell (BioRad) transfer apparatus.

    Techniques: Purification, Polyacrylamide Gel Electrophoresis, Western Blot

    The over-expression of RALDH1 resulted in the RAL-mediated induction of Srebp-1c in 833/15 INS-1 cells. A. The adenovirus-mediated Raldh1 mRNA expression. B. Immuno-blot of the over-expression of RALDH1 protein in INS-1 cells. Whole cell lysates (50 µg/sample) of the control cells (lane 1), cells infected by the indicated pfu of Ad-β-gal (lane 2) or Ad-Raldh1 (lanes 3–5) were separated in 8% SDS protein gels, and transferred to the PVDF membranes. Primary antibodies to RALDH1 (1∶1000 dilution in TBST containing 5% dry milk), and to β-Actin (1∶1000 dilution in TBST containing 5% bovine serum albumin) were recognized by goat anti-rabbit IgG conjugated to horseradish peroxidase, and visualized by chemiluminescence. The films were scanned and presented as described in the Material and Methods . C. RAL only induced Srebp-1c expression in cells over-expressing RALDH1, but not β-gal. D. RA induced Srebp-1c expression in cells over-expressing either β-gal or RALDH1. Results were presented as means ± SD of fold inductions (* for comparing the different dosages of RAL in cells infected by Ad-Raldh1 using one way ANOVA, n = 3, all p

    Journal: PLoS ONE

    Article Title: The Hepatic Raldh1 Expression Is elevated in Zucker Fatty Rats and Its Over-Expression Introduced the Retinal-Induced Srebp-1c Expression in INS-1 Cells

    doi: 10.1371/journal.pone.0045210

    Figure Lengend Snippet: The over-expression of RALDH1 resulted in the RAL-mediated induction of Srebp-1c in 833/15 INS-1 cells. A. The adenovirus-mediated Raldh1 mRNA expression. B. Immuno-blot of the over-expression of RALDH1 protein in INS-1 cells. Whole cell lysates (50 µg/sample) of the control cells (lane 1), cells infected by the indicated pfu of Ad-β-gal (lane 2) or Ad-Raldh1 (lanes 3–5) were separated in 8% SDS protein gels, and transferred to the PVDF membranes. Primary antibodies to RALDH1 (1∶1000 dilution in TBST containing 5% dry milk), and to β-Actin (1∶1000 dilution in TBST containing 5% bovine serum albumin) were recognized by goat anti-rabbit IgG conjugated to horseradish peroxidase, and visualized by chemiluminescence. The films were scanned and presented as described in the Material and Methods . C. RAL only induced Srebp-1c expression in cells over-expressing RALDH1, but not β-gal. D. RA induced Srebp-1c expression in cells over-expressing either β-gal or RALDH1. Results were presented as means ± SD of fold inductions (* for comparing the different dosages of RAL in cells infected by Ad-Raldh1 using one way ANOVA, n = 3, all p

    Article Snippet: Proteins (40 μg/lane) in whole cell lysates were separated on SDS-PAGE, transferred to BIO-RAD Immuno-Blot PVDF membrane (Hercules, CA) and detected with primary antibodies to RALDH1 (for , catalog #2052-1, Epitomics, Burlingame, CA 94010), RALDH1 (for , catalog #AP1465a, Abgent, San Diego, CA 92121), CYP26A1 (catalog # CYP26A11-A, Alpha Diagnostics International, TX 78244), and β-Actin (#4970 s, Cell Signaling Technology, Danvers, MA 01923) according to the protocols provided by the manufacturers.

    Techniques: Over Expression, Expressing, Infection

    Deletion of the C-terminal domain increases auto-phosphorylation activity. ( A ) Partial tryptic digestion of recombinant MPK10. 50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at the indicated time points and the reaction was stopped either by adding Laemmli buffer (for N-terminal sequencing) or by lowering the pH to 5.0 and subsequent freezing (for mass determination by SELDI-TOF). For N-terminal sequencing, samples were separated by SDS-PAGE, transferred on PVDF membrane and stained by amidoblack. N-terminal sequencing was performed at the protein analysis platform at the Institut Pasteur. For mass determination, samples were immobilized on a H4 ProteinChip Array (C16 reversed phase surface) and peptide masses identified by SELDI-TOF. Results of the N-terminal sequencing are represented by the cartoon in ( B ), and the sequences are indicated in ( C ). Italic characters represent the Strep3-tag and bold characters represent the sequence of Leishmania major MPK10. White and grey arrowheads indicate respectively lysine or arginine residues recognized by trypsine, including K12, K24, K30 and R392. The white arrow at the position D387 indicates the position of the last cleaved residue resulting in the generation of the form lacking the last 46 amino acids of MPK10. ( D ) In vitro kinase assay using recombinant His-MPK10 (NM) and the truncated kinase mutants His-MPK10-ΔC (ΔC), and His-MPK10-ΔC_K51A (ΔC_K/A). Results are representative of three independent experiments. Purified proteins were incubated with four different substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed at the same time for 30 min at pH 7.5 and 37°C and reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (right), and signals were revealed by auto-radiography with the same exposure time between the different gels (left). The brackets in (D) indicate auto-phosphorylation (Auto-P) and substrate phosphorylation (Substrate-P) signals.

    Journal: PLoS Pathogens

    Article Title: Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

    doi: 10.1371/journal.ppat.1004347

    Figure Lengend Snippet: Deletion of the C-terminal domain increases auto-phosphorylation activity. ( A ) Partial tryptic digestion of recombinant MPK10. 50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at the indicated time points and the reaction was stopped either by adding Laemmli buffer (for N-terminal sequencing) or by lowering the pH to 5.0 and subsequent freezing (for mass determination by SELDI-TOF). For N-terminal sequencing, samples were separated by SDS-PAGE, transferred on PVDF membrane and stained by amidoblack. N-terminal sequencing was performed at the protein analysis platform at the Institut Pasteur. For mass determination, samples were immobilized on a H4 ProteinChip Array (C16 reversed phase surface) and peptide masses identified by SELDI-TOF. Results of the N-terminal sequencing are represented by the cartoon in ( B ), and the sequences are indicated in ( C ). Italic characters represent the Strep3-tag and bold characters represent the sequence of Leishmania major MPK10. White and grey arrowheads indicate respectively lysine or arginine residues recognized by trypsine, including K12, K24, K30 and R392. The white arrow at the position D387 indicates the position of the last cleaved residue resulting in the generation of the form lacking the last 46 amino acids of MPK10. ( D ) In vitro kinase assay using recombinant His-MPK10 (NM) and the truncated kinase mutants His-MPK10-ΔC (ΔC), and His-MPK10-ΔC_K51A (ΔC_K/A). Results are representative of three independent experiments. Purified proteins were incubated with four different substrates, including 12 µg of histone H1, 9 µg of Ets1, 36 µg of casein, and 25 µg of MBP. Recombinant human MEK1 was used as positive control with MBP as substrate. Kinase assays were performed at the same time for 30 min at pH 7.5 and 37°C and reaction samples were separated by SDS-PAGE, gels were stained by Coomassie (right), and signals were revealed by auto-radiography with the same exposure time between the different gels (left). The brackets in (D) indicate auto-phosphorylation (Auto-P) and substrate phosphorylation (Substrate-P) signals.

    Article Snippet: Alternatively, proteins were separated by SDS–PAGE on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and blotted onto polyvinylidene difluoride (PVDF) membranes (Pierce).

    Techniques: Activity Assay, Recombinant, Sequencing, SDS Page, Staining, In Vitro, Kinase Assay, Purification, Incubation, Positive Control

    Levels of haptoglobin and SAP but not hemopexin, transferrin, or CRP are elevated in the plasma of ASGP-R2(-/-) mice. Equal amounts of plasma protein per sample, 1 μg for hemopexin, 10 μg for haptoglobin, transferrin, and CRP or 100 μg for SAP detection, from 7 Wt and 11 ASGP-R2(-/-) mice were separated by SDS-PAGE and electrophoretically transferred to Immobilon-FL PVDF membranes. Western blots were performed to quantitate the relative amount of: ( panel A ) haptoglobin, ( panel B ) SAP, ( panel C ) hemopexin, ( panel D ) transferrin, and ( panel E ) CRP in each sample. Haptoglobin is elevated 4-fold, p = 0.01, and SAP 2-fold, p = 0.01 in plasma from ASGP-R2(-/-) mice. Hemopexin, transferrin, and CRP did not differ significantly in levels in Wt and ASGP-R2(-/-) mice.

    Journal: The Journal of Biological Chemistry

    Article Title: The Asialoglycoprotein Receptor Regulates Levels of Plasma Glycoproteins Terminating with Sialic Acid α2,6-Galactose

    doi: 10.1074/jbc.M808689200

    Figure Lengend Snippet: Levels of haptoglobin and SAP but not hemopexin, transferrin, or CRP are elevated in the plasma of ASGP-R2(-/-) mice. Equal amounts of plasma protein per sample, 1 μg for hemopexin, 10 μg for haptoglobin, transferrin, and CRP or 100 μg for SAP detection, from 7 Wt and 11 ASGP-R2(-/-) mice were separated by SDS-PAGE and electrophoretically transferred to Immobilon-FL PVDF membranes. Western blots were performed to quantitate the relative amount of: ( panel A ) haptoglobin, ( panel B ) SAP, ( panel C ) hemopexin, ( panel D ) transferrin, and ( panel E ) CRP in each sample. Haptoglobin is elevated 4-fold, p = 0.01, and SAP 2-fold, p = 0.01 in plasma from ASGP-R2(-/-) mice. Hemopexin, transferrin, and CRP did not differ significantly in levels in Wt and ASGP-R2(-/-) mice.

    Article Snippet: Plasma proteins from individual Wt and ASGP-R2(-/-) mice were separated by SDS-PAGE and electrophoretically transferred to Immobilon-FL PVDF membranes (Millipore).

    Techniques: Mouse Assay, SDS Page, Western Blot

    Haptoglobin-bearing oligosaccharides that terminate with Siaα2,6Gal predominate in the plasma of Wt and ASGP-R2(-/-). Plasma, 25 μg, from Wt and ASGP-R2(-/-) mice was incubated with SNA-I-agarose ( panel A ) or RCA-I-agarose ( panel B ). Equal aliquots of the supernatant containing unbound haptoglobin, each of three wash fractions, and material eluted by warming the agarose in SDS-PAGE-loading buffer was subjected to SDS-PAGE and electrophoretic transfer to Immobilon-F PVDF membranes. The amount of haptoglobin in each fraction was determined by Western blotting using rabbit anti-mouse haptoglobin. The amount of haptoglobin in each fraction is shown as a percent of the total.

    Journal: The Journal of Biological Chemistry

    Article Title: The Asialoglycoprotein Receptor Regulates Levels of Plasma Glycoproteins Terminating with Sialic Acid α2,6-Galactose

    doi: 10.1074/jbc.M808689200

    Figure Lengend Snippet: Haptoglobin-bearing oligosaccharides that terminate with Siaα2,6Gal predominate in the plasma of Wt and ASGP-R2(-/-). Plasma, 25 μg, from Wt and ASGP-R2(-/-) mice was incubated with SNA-I-agarose ( panel A ) or RCA-I-agarose ( panel B ). Equal aliquots of the supernatant containing unbound haptoglobin, each of three wash fractions, and material eluted by warming the agarose in SDS-PAGE-loading buffer was subjected to SDS-PAGE and electrophoretic transfer to Immobilon-F PVDF membranes. The amount of haptoglobin in each fraction was determined by Western blotting using rabbit anti-mouse haptoglobin. The amount of haptoglobin in each fraction is shown as a percent of the total.

    Article Snippet: Plasma proteins from individual Wt and ASGP-R2(-/-) mice were separated by SDS-PAGE and electrophoretically transferred to Immobilon-FL PVDF membranes (Millipore).

    Techniques: Mouse Assay, Incubation, SDS Page, Western Blot

    Glycoproteins bearing terminal Siaα2,6Galβ1,4GlcNAc are elevated in the plasma of ASGP-R2(-/-) mice. Equal amounts of Cy5-labeled plasma proteins, 75 μg from Wt ( panel A ) and ASGP-R2(-/-) ( panel B ) mice, respectively, were separately subjected to 2D-GE and electrophoretically transferred to Immobilon-FL PVDF membranes. The membranes were then probed with HRP-SNA-I, which recognizes structures terminating with Siaα2,6Galβ1,4GlcNAc. All spots except one that were identified with an increase in ASGP-R2(-/-) mice corresponded to glycoproteins that are recognized by SNA-I and show increased reactivity in plasma from ASGP-R2(-/-) mice. Compare circled regions in panels A and B .

    Journal: The Journal of Biological Chemistry

    Article Title: The Asialoglycoprotein Receptor Regulates Levels of Plasma Glycoproteins Terminating with Sialic Acid α2,6-Galactose

    doi: 10.1074/jbc.M808689200

    Figure Lengend Snippet: Glycoproteins bearing terminal Siaα2,6Galβ1,4GlcNAc are elevated in the plasma of ASGP-R2(-/-) mice. Equal amounts of Cy5-labeled plasma proteins, 75 μg from Wt ( panel A ) and ASGP-R2(-/-) ( panel B ) mice, respectively, were separately subjected to 2D-GE and electrophoretically transferred to Immobilon-FL PVDF membranes. The membranes were then probed with HRP-SNA-I, which recognizes structures terminating with Siaα2,6Galβ1,4GlcNAc. All spots except one that were identified with an increase in ASGP-R2(-/-) mice corresponded to glycoproteins that are recognized by SNA-I and show increased reactivity in plasma from ASGP-R2(-/-) mice. Compare circled regions in panels A and B .

    Article Snippet: Plasma proteins from individual Wt and ASGP-R2(-/-) mice were separated by SDS-PAGE and electrophoretically transferred to Immobilon-FL PVDF membranes (Millipore).

    Techniques: Mouse Assay, Labeling

    Dot blotting analysis for binding site confirmation. Dot blotting was performed to define the geometric orientations of the selected aptamers. ( a ) LCN2 (74.2 pmol) with 9 selected aptamers (LCN2_Apta1 to LCN2_Apta9, 742 pmol) and controls (C1 to C4) were dotted onto the Hybond-P PVDF membrane. After incubation with the HRP-conjugated anti-LCN2 polyclonal antibody, an assay image was taken using an ECL assay protocol. The plotted signal intensities were calculated using ImageJ software and normalized to C2 (LCN2 74.2 pmol). The schematic epitope binding of two aptamers (LCN2_apta2 and LCN2_apta4) is illustrated in ( b ). All parts of this figure were drawn by the authors K-A. L. and J-Y. A.

    Journal: Scientific Reports

    Article Title: Aptamer-based Sandwich Assay and its Clinical Outlooks for Detecting Lipocalin-2 in Hepatocellular Carcinoma (HCC)

    doi: 10.1038/srep10897

    Figure Lengend Snippet: Dot blotting analysis for binding site confirmation. Dot blotting was performed to define the geometric orientations of the selected aptamers. ( a ) LCN2 (74.2 pmol) with 9 selected aptamers (LCN2_Apta1 to LCN2_Apta9, 742 pmol) and controls (C1 to C4) were dotted onto the Hybond-P PVDF membrane. After incubation with the HRP-conjugated anti-LCN2 polyclonal antibody, an assay image was taken using an ECL assay protocol. The plotted signal intensities were calculated using ImageJ software and normalized to C2 (LCN2 74.2 pmol). The schematic epitope binding of two aptamers (LCN2_apta2 and LCN2_apta4) is illustrated in ( b ). All parts of this figure were drawn by the authors K-A. L. and J-Y. A.

    Article Snippet: Then, 1 μL of each aptamer-LCN2 complex was spotted onto a Hybond-P PVDF membrane (GE Healthcare, Uppsala, Sweden).

    Techniques: Binding Assay, Incubation, Software

    Purification of AtAPY1 and AtAPY1-δTM. The explanation of the colors in the schematic representations of AtAPY1 and AtAPY1-δTM can be found in Fig. 2 . (A) Total proteins from 1.6 x 10 5 HEK293 cells were harvested at each of the indicated time points post transfection with AtAPY1 DNA, separated in a 4–12% gradient gel under denaturing conditions, transferred onto a PVDF membrane and successively incubated with anti-APY1 and anti-His antibodies (left panel). The black arrows mark the signal specific for AtAPY1, while the gray arrowheads indicate unspecific bands. The right panel shows the total protein extract from 1.4 x 10 8 HEK293 cells harvested at 89 h after transfection with AtAPY1 DNA subjected to Ni 2+ -affinity chromatography. Various fractions were separated in a 4–12% gel under denaturing conditions and either stained with Coomassie or transferred onto a PVDF membrane for Western blot analysis. The black arrow indicates the signal detected with antibodies against AtAPY1. The volumes loaded were 1/480 of the flow through (FT) fraction, 1/50 of each of the final two wash fractions W3 and W4 and 1/100 of the elution fraction E. (B) The left panel shows samples representing equal volumes (1/3,000) of the culture medium of 1 x 10 8 HEK293 cells taken at the indicated time points post transfection with AtAPY1-δTM DNA and separated in a 4–12% gradient gel under denaturing conditions. Subsequently, the proteins were either stained with Coomassie or blotted onto a PVDF membrane for Western blot analysis. The right panel depicts the culture medium of 4 x 10 7 HEK293 cells at time point 88 h after transfection with AtAPY1-δTM DNA subjected to Ni 2+ -affinity chromatography. A gradient gel (4–12%) was loaded with 20 μL of supernatant (S) and 20 μL of flow through (FT), 10 μL of each wash 1–5 and 10 μL of each eluate 1–2. For total volumes of the individual fractions see Materials and Methods . The protein amount loaded for eluate 1 equals about 70 ng. Following SDS-PAGE, the gel was silver-stained.

    Journal: PLoS ONE

    Article Title: The Biochemical Properties of the Arabidopsis Ecto-Nucleoside Triphosphate Diphosphohydrolase AtAPY1 Contradict a Direct Role in Purinergic Signaling

    doi: 10.1371/journal.pone.0115832

    Figure Lengend Snippet: Purification of AtAPY1 and AtAPY1-δTM. The explanation of the colors in the schematic representations of AtAPY1 and AtAPY1-δTM can be found in Fig. 2 . (A) Total proteins from 1.6 x 10 5 HEK293 cells were harvested at each of the indicated time points post transfection with AtAPY1 DNA, separated in a 4–12% gradient gel under denaturing conditions, transferred onto a PVDF membrane and successively incubated with anti-APY1 and anti-His antibodies (left panel). The black arrows mark the signal specific for AtAPY1, while the gray arrowheads indicate unspecific bands. The right panel shows the total protein extract from 1.4 x 10 8 HEK293 cells harvested at 89 h after transfection with AtAPY1 DNA subjected to Ni 2+ -affinity chromatography. Various fractions were separated in a 4–12% gel under denaturing conditions and either stained with Coomassie or transferred onto a PVDF membrane for Western blot analysis. The black arrow indicates the signal detected with antibodies against AtAPY1. The volumes loaded were 1/480 of the flow through (FT) fraction, 1/50 of each of the final two wash fractions W3 and W4 and 1/100 of the elution fraction E. (B) The left panel shows samples representing equal volumes (1/3,000) of the culture medium of 1 x 10 8 HEK293 cells taken at the indicated time points post transfection with AtAPY1-δTM DNA and separated in a 4–12% gradient gel under denaturing conditions. Subsequently, the proteins were either stained with Coomassie or blotted onto a PVDF membrane for Western blot analysis. The right panel depicts the culture medium of 4 x 10 7 HEK293 cells at time point 88 h after transfection with AtAPY1-δTM DNA subjected to Ni 2+ -affinity chromatography. A gradient gel (4–12%) was loaded with 20 μL of supernatant (S) and 20 μL of flow through (FT), 10 μL of each wash 1–5 and 10 μL of each eluate 1–2. For total volumes of the individual fractions see Materials and Methods . The protein amount loaded for eluate 1 equals about 70 ng. Following SDS-PAGE, the gel was silver-stained.

    Article Snippet: Immunoblotting Protein samples were separated by SDS-PAGE and transferred to BioTrace NT nitrocellulose or 0.45-μm PVDF membranes (both from PALL Life Sciences).

    Techniques: Purification, Transfection, Incubation, Affinity Chromatography, Staining, Western Blot, Flow Cytometry, SDS Page

    Total membrane fractions of Chara internodal cells. (A) Proteins of membrane fractions (MF) were separated by SDS-PAGE (10%), stained with Coomassie Brilliant Blue (CBB) or blotted onto PVDF membranes for immunodetection of the plasma membrane H + ATPase. 30 μg protein per lane. Numbers on the left refer to molecular weight markers in kDa. Only the upper part of the PVDF membrane was used, the lower part was probed for immunodetection of low molecular weight proteins. (B) Immunodetection of selected organelle marker proteins for vacuoles (VHA-ɛ, H + PPase), ER (BiP2), plasma membrane and endosomal compartments (ARA6) or cytosol (tubulin, GRF 14-3-3). Proteins of the MFs were separated by preparative SDS-PAGE (12.75% or 10% for GRF and ARA6), plotted onto PVDF membranes cut into 3 mm strips and detected with the respective antibodies. 10 μg protein per strip. Molecular weight markers are given in kDa. Arrow heads indicate the expected position of the respective protein.

    Journal: PLoS ONE

    Article Title: Dissecting the subcellular membrane proteome reveals enrichment of H+ (co-)transporters and vesicle trafficking proteins in acidic zones of Chara internodal cells

    doi: 10.1371/journal.pone.0201480

    Figure Lengend Snippet: Total membrane fractions of Chara internodal cells. (A) Proteins of membrane fractions (MF) were separated by SDS-PAGE (10%), stained with Coomassie Brilliant Blue (CBB) or blotted onto PVDF membranes for immunodetection of the plasma membrane H + ATPase. 30 μg protein per lane. Numbers on the left refer to molecular weight markers in kDa. Only the upper part of the PVDF membrane was used, the lower part was probed for immunodetection of low molecular weight proteins. (B) Immunodetection of selected organelle marker proteins for vacuoles (VHA-ɛ, H + PPase), ER (BiP2), plasma membrane and endosomal compartments (ARA6) or cytosol (tubulin, GRF 14-3-3). Proteins of the MFs were separated by preparative SDS-PAGE (12.75% or 10% for GRF and ARA6), plotted onto PVDF membranes cut into 3 mm strips and detected with the respective antibodies. 10 μg protein per strip. Molecular weight markers are given in kDa. Arrow heads indicate the expected position of the respective protein.

    Article Snippet: For immunodetection, separated proteins were transferred onto PVDF membranes (Roth, Karlsruhe, Germany) by electro-transfer with 20 V for 1 h (Semi Dry Electrophoretic Transfer Cell, Bio-Rad, Vienna, Austria).

    Techniques: SDS Page, Staining, Immunodetection, Molecular Weight, Marker, Stripping Membranes

    Differences in charasome abundance and protein expression in alkaline and acidic regions of Chara cells. (A) Left image pair: FM1-43-labelled charasomes (green fluorescence) and chloroplasts (bright field image) at an acidic band. Right image pair: FM1-43-stained charasomes are absent from the alkaline band; the bright field images show the chloroplasts. An FM1-43-stained internodal cell was cut into acid and alkaline regions as described in Materials and Methods guided by pH banding pattern visualized by phenol red. Cell fragments were mounted in artificial fresh water and examined in the CLSM. Bar = 20 μm (B) Proteins of membrane fractions (MF) and cytosolic fractions (CF) obtained from acidic (ac) and alkaline (alk) regions were separated by SDS-PAGE (10%) and stained with Comassie Brilliant Blue (CBB) or blotted onto PVDF membranes for immunodetection of the plasma membrane H + ATPase using only the upper part of the membrane. 15 μg protein was loaded per lane. Numbers on the left refer to molecular weight markers in kDa. (C) Immunodetection of selected higher and lower molecular weight proteins from the membrane fraction (MF) as indicated. 10 μg protein was loaded per lane, 10% gel. PVDF membrane was cut into upper and lower halves at 50 kDa. Molecular weight markers given in kDa.

    Journal: PLoS ONE

    Article Title: Dissecting the subcellular membrane proteome reveals enrichment of H+ (co-)transporters and vesicle trafficking proteins in acidic zones of Chara internodal cells

    doi: 10.1371/journal.pone.0201480

    Figure Lengend Snippet: Differences in charasome abundance and protein expression in alkaline and acidic regions of Chara cells. (A) Left image pair: FM1-43-labelled charasomes (green fluorescence) and chloroplasts (bright field image) at an acidic band. Right image pair: FM1-43-stained charasomes are absent from the alkaline band; the bright field images show the chloroplasts. An FM1-43-stained internodal cell was cut into acid and alkaline regions as described in Materials and Methods guided by pH banding pattern visualized by phenol red. Cell fragments were mounted in artificial fresh water and examined in the CLSM. Bar = 20 μm (B) Proteins of membrane fractions (MF) and cytosolic fractions (CF) obtained from acidic (ac) and alkaline (alk) regions were separated by SDS-PAGE (10%) and stained with Comassie Brilliant Blue (CBB) or blotted onto PVDF membranes for immunodetection of the plasma membrane H + ATPase using only the upper part of the membrane. 15 μg protein was loaded per lane. Numbers on the left refer to molecular weight markers in kDa. (C) Immunodetection of selected higher and lower molecular weight proteins from the membrane fraction (MF) as indicated. 10 μg protein was loaded per lane, 10% gel. PVDF membrane was cut into upper and lower halves at 50 kDa. Molecular weight markers given in kDa.

    Article Snippet: For immunodetection, separated proteins were transferred onto PVDF membranes (Roth, Karlsruhe, Germany) by electro-transfer with 20 V for 1 h (Semi Dry Electrophoretic Transfer Cell, Bio-Rad, Vienna, Austria).

    Techniques: Expressing, Fluorescence, Staining, Confocal Laser Scanning Microscopy, SDS Page, Immunodetection, Molecular Weight

    The Aptamers of F5R1 and F5R2 Enhanced Lysosomal Degradation of α-syn and Rescued the Cell Defects (A) SK-N-SH cells pre-treated with F5R1, F5R2 or random DNA sequence were transfected the α-syn or vector control vectors and incubated for 24 hr. The extracts were separated by SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked and probed with the α-syn specific polyclonal antibody. β-actin served as the loading control. (B) Quantitative analysis of the total protein level of α-syn from (A). (C) SK-N-SH cells were similarly treated as in (A) except for incubation time (48 hr). The cell extracts were immunoblotted with the α-syn polyclonal antibody. β-actin served as the loading control. (D) Quantitative analysis of the total protein level of α-syn from (C). Data are presented as the mean ± SD (one-way ANOVA) ***p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

    doi: 10.1016/j.omtn.2018.02.011

    Figure Lengend Snippet: The Aptamers of F5R1 and F5R2 Enhanced Lysosomal Degradation of α-syn and Rescued the Cell Defects (A) SK-N-SH cells pre-treated with F5R1, F5R2 or random DNA sequence were transfected the α-syn or vector control vectors and incubated for 24 hr. The extracts were separated by SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked and probed with the α-syn specific polyclonal antibody. β-actin served as the loading control. (B) Quantitative analysis of the total protein level of α-syn from (A). (C) SK-N-SH cells were similarly treated as in (A) except for incubation time (48 hr). The cell extracts were immunoblotted with the α-syn polyclonal antibody. β-actin served as the loading control. (D) Quantitative analysis of the total protein level of α-syn from (C). Data are presented as the mean ± SD (one-way ANOVA) ***p

    Article Snippet: Proteins were resolved by 10% SDS-PAGE and transferred to a PVDF membrane, which was blocked and then incubated with anti-α-syn (BD Biosciences, Franklin Lakes, NJ), anti-β-actin (Beijing Guanxingyu, China), and anti-VDAC1 (Abcam, Cambridge, UK) for 2 hr at room temperature.

    Techniques: Sequencing, Transfection, Plasmid Preparation, Incubation, SDS Page