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  • 99
    Thermo Fisher pvdf membrane
    Pvdf Membrane, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore polyvinylidene difluoride membranes
    Polyvinylidene Difluoride Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 35088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad pvdf membrane
    Pvdf Membrane, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 30039 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore polyvinylidene difluoride pvdf membranes
    Polyvinylidene Difluoride Pvdf Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15823 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad polyvinylidene difluoride pvdf membrane
    Polyvinylidene Difluoride Pvdf Membrane, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 7102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Merck KGaA pvdf membrane
    Protein modification by PGA 1 -biotin and [1- 14 C]OPDA. ( A ) Proteins were extracted from leaves and incubated with (+) or without (-) PGA 1 -biotin [110 μM]. Protein samples (120 μg) were separated on a 10% <t>SDS-PAGE</t> and transferred to a <t>PVDF</t> membrane. Biotinylated proteins were detected by NeutrAvidin-HRP (left panel) followed by protein staining with Coomassie (loading control, right panel). ( B ) Leaf discs were exposed to [1- 14 C]OPDA for 2 h. Crude extract was centrifuged and an aliquot of the supernatant was analyzed by SDS-PAGE followed by radiography.
    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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    94
    Santa Cruz Biotechnology pvdf membrane
    SerpinB5 phosphorylation in the lactating mammary gland. A. 100 ug of protein extracts from mammary gland isolated on days 1, 10 and 20 of lactation (1L, 10L and 20L), and on days 1 and 5 of involution (1I and 5I) were resolved by 8% Phos-Tag™ <t>SDS-PAGE</t> gel. Proteins were transferred to <t>PVDF</t> membrane and probed with rabbit anti-SerpinB5 (Sigma). Arrows indicate two phosphorylated SerpinB5 bands (P- SerpinB5-1 and P- SerpinB5-2) and arrowhead indicates unphosphorylated SerpinB5. Top and middle panels represent a short and a long X-ray film exposure, respectively. The membrane was reincubated with anti-HSP90 for a loading control; B. each of the three bands was quantified and expressed as percentage of the total levels of detected SerpinB5. This result is representative of two independent experiments; C. 2 mg of protein extracts of mammary gland isolated on days 10 and 20 of lactation were analyzed by 2D-SDS-PAGE using 17 cm pH 4.7–5.9 IPG strips followed by Western blot with anti-SerpinB5 (Santa Cruz). This result is representative of two independent experiments.
    Pvdf Membrane, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 4313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam pvdf membranes
    SerpinB5 phosphorylation in the lactating mammary gland. A. 100 ug of protein extracts from mammary gland isolated on days 1, 10 and 20 of lactation (1L, 10L and 20L), and on days 1 and 5 of involution (1I and 5I) were resolved by 8% Phos-Tag™ <t>SDS-PAGE</t> gel. Proteins were transferred to <t>PVDF</t> membrane and probed with rabbit anti-SerpinB5 (Sigma). Arrows indicate two phosphorylated SerpinB5 bands (P- SerpinB5-1 and P- SerpinB5-2) and arrowhead indicates unphosphorylated SerpinB5. Top and middle panels represent a short and a long X-ray film exposure, respectively. The membrane was reincubated with anti-HSP90 for a loading control; B. each of the three bands was quantified and expressed as percentage of the total levels of detected SerpinB5. This result is representative of two independent experiments; C. 2 mg of protein extracts of mammary gland isolated on days 10 and 20 of lactation were analyzed by 2D-SDS-PAGE using 17 cm pH 4.7–5.9 IPG strips followed by Western blot with anti-SerpinB5 (Santa Cruz). This result is representative of two independent experiments.
    Pvdf Membranes, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 5076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc pvdf membranes
    Derivatives of Yersinia pestis KIM8-3002 (KIM5 pPCP1-minus, Sm r ) were grown in a chemically defined medium at 26°C for 2 h in the presence (lanes 1, 3, and 5) or absence of calcium (lanes 2, 4, and 6). Lanes 1 and 2 contain Y. pestis KIM8-3002. Lanes 3 and 4 contain Y. pestis KIM8-3002 Δ yscF expressing YscF from pBAD18-YscF. Lanes 5 and 6 contain Y. pestis KIM8-3002 Δ yscF gene. After the 2 h growth, the culture was shifted to 37°C to induce expression of the Ysc type III secretion system and the Low Calcium Response. Following 4 h of growth at 37°C cultures, were centrifuged to obtain whole cell fractions and cell-free culture supernatant fractions. Total proteins from each fraction were precipitated with 10% trichloro acetic acid. Dried proteins were resuspended in <t>SDS-PAGE</t> sample buffer and electrophoresed in a 15% SDS-PAGE gel. Proteins were then transferred to a <t>PVDF</t> membrane and immuno-blotted with pooled mouse serum used at a 1:20,000 dilution. Mouse serum was obtained by bleeding mice subsequent to immunization with HT-YscF, serum from several mice was pooled to control for animal specific variation. The position and sizes for the molecular weight markers are indicated and the position of YscF is shown.
    Pvdf Membranes, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 5078 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore immobilon polyvinylidene difluoride membranes
    Derivatives of Yersinia pestis KIM8-3002 (KIM5 pPCP1-minus, Sm r ) were grown in a chemically defined medium at 26°C for 2 h in the presence (lanes 1, 3, and 5) or absence of calcium (lanes 2, 4, and 6). Lanes 1 and 2 contain Y. pestis KIM8-3002. Lanes 3 and 4 contain Y. pestis KIM8-3002 Δ yscF expressing YscF from pBAD18-YscF. Lanes 5 and 6 contain Y. pestis KIM8-3002 Δ yscF gene. After the 2 h growth, the culture was shifted to 37°C to induce expression of the Ysc type III secretion system and the Low Calcium Response. Following 4 h of growth at 37°C cultures, were centrifuged to obtain whole cell fractions and cell-free culture supernatant fractions. Total proteins from each fraction were precipitated with 10% trichloro acetic acid. Dried proteins were resuspended in <t>SDS-PAGE</t> sample buffer and electrophoresed in a 15% SDS-PAGE gel. Proteins were then transferred to a <t>PVDF</t> membrane and immuno-blotted with pooled mouse serum used at a 1:20,000 dilution. Mouse serum was obtained by bleeding mice subsequent to immunization with HT-YscF, serum from several mice was pooled to control for animal specific variation. The position and sizes for the molecular weight markers are indicated and the position of YscF is shown.
    Immobilon Polyvinylidene Difluoride Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 848 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher tropifluor pvdf membrane pore size
    Derivatives of Yersinia pestis KIM8-3002 (KIM5 pPCP1-minus, Sm r ) were grown in a chemically defined medium at 26°C for 2 h in the presence (lanes 1, 3, and 5) or absence of calcium (lanes 2, 4, and 6). Lanes 1 and 2 contain Y. pestis KIM8-3002. Lanes 3 and 4 contain Y. pestis KIM8-3002 Δ yscF expressing YscF from pBAD18-YscF. Lanes 5 and 6 contain Y. pestis KIM8-3002 Δ yscF gene. After the 2 h growth, the culture was shifted to 37°C to induce expression of the Ysc type III secretion system and the Low Calcium Response. Following 4 h of growth at 37°C cultures, were centrifuged to obtain whole cell fractions and cell-free culture supernatant fractions. Total proteins from each fraction were precipitated with 10% trichloro acetic acid. Dried proteins were resuspended in <t>SDS-PAGE</t> sample buffer and electrophoresed in a 15% SDS-PAGE gel. Proteins were then transferred to a <t>PVDF</t> membrane and immuno-blotted with pooled mouse serum used at a 1:20,000 dilution. Mouse serum was obtained by bleeding mice subsequent to immunization with HT-YscF, serum from several mice was pooled to control for animal specific variation. The position and sizes for the molecular weight markers are indicated and the position of YscF is shown.
    Tropifluor Pvdf Membrane Pore Size, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore immobilon fl pvdf membranes
    Derivatives of Yersinia pestis KIM8-3002 (KIM5 pPCP1-minus, Sm r ) were grown in a chemically defined medium at 26°C for 2 h in the presence (lanes 1, 3, and 5) or absence of calcium (lanes 2, 4, and 6). Lanes 1 and 2 contain Y. pestis KIM8-3002. Lanes 3 and 4 contain Y. pestis KIM8-3002 Δ yscF expressing YscF from pBAD18-YscF. Lanes 5 and 6 contain Y. pestis KIM8-3002 Δ yscF gene. After the 2 h growth, the culture was shifted to 37°C to induce expression of the Ysc type III secretion system and the Low Calcium Response. Following 4 h of growth at 37°C cultures, were centrifuged to obtain whole cell fractions and cell-free culture supernatant fractions. Total proteins from each fraction were precipitated with 10% trichloro acetic acid. Dried proteins were resuspended in <t>SDS-PAGE</t> sample buffer and electrophoresed in a 15% SDS-PAGE gel. Proteins were then transferred to a <t>PVDF</t> membrane and immuno-blotted with pooled mouse serum used at a 1:20,000 dilution. Mouse serum was obtained by bleeding mice subsequent to immunization with HT-YscF, serum from several mice was pooled to control for animal specific variation. The position and sizes for the molecular weight markers are indicated and the position of YscF is shown.
    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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    93
    Pall Corporation pvdf membrane
    hnRNP E1 but not hnRNP E2 inhibit expression of subgenomic HIV-1 expression vectors . (A) Schematic of HIV-1 env expression vector, pgTat, including the location of the ESE and ESS. The sequences of pgTat derivatives pgTatΔESE and pgTatΔESEΔESS SL1 are shown. (B) HeLa cells were co-transfected with pgTat or derivatives thereof, SVH6Rev, and hnRNP E expressing plasmids as indicated. 48 hours post-transfection, cells were harvested in RIPA buffer, and lysates fractionated on SDS-PAGE gels. Following transfer to <t>PVDF</t> membranes, blots were probed with antibody against gp120 and tubulin. (C) Effect of hnRNP E proteins on Gag/RRE expression. Shown is a diagram of the Gag expression construct used. HeLa cells were transfected with Gag/RRE, hnRNP E and Rev expression constructs as indicated. 48 hrs post transfection, cells were harvested, and lysates fractionated on SDS-PAGE gels. Blots were probed with antibody against Rev and <t>p24,</t> stripped then reprobed for tubulin and myc to confirm equal loading and similar expression of myc-tagged constructs respectively. To quantitate changes in Rev protein expression, blots were scanned and analyzed in Imagequant. Summary of 5 independent determinations is shown (D). (E) Effect of hnRNP E proteins on SEAP expression. Media from cells transfected with a SEAP expressing plasmid was harvested 48 hrs post transfection and SEAP expression normalized to that seen upon cotransfection with the CMVmyc control vector. Results shown are an average of 3 experiments. Error bars show standard deviation. SEAP expression of the myc control vector was set to 1.
    Pvdf Membrane, supplied by Pall Corporation, used in various techniques. Bioz Stars score: 93/100, based on 1090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Beyotime pvdf membrane
    hnRNP E1 but not hnRNP E2 inhibit expression of subgenomic HIV-1 expression vectors . (A) Schematic of HIV-1 env expression vector, pgTat, including the location of the ESE and ESS. The sequences of pgTat derivatives pgTatΔESE and pgTatΔESEΔESS SL1 are shown. (B) HeLa cells were co-transfected with pgTat or derivatives thereof, SVH6Rev, and hnRNP E expressing plasmids as indicated. 48 hours post-transfection, cells were harvested in RIPA buffer, and lysates fractionated on SDS-PAGE gels. Following transfer to <t>PVDF</t> membranes, blots were probed with antibody against gp120 and tubulin. (C) Effect of hnRNP E proteins on Gag/RRE expression. Shown is a diagram of the Gag expression construct used. HeLa cells were transfected with Gag/RRE, hnRNP E and Rev expression constructs as indicated. 48 hrs post transfection, cells were harvested, and lysates fractionated on SDS-PAGE gels. Blots were probed with antibody against Rev and <t>p24,</t> stripped then reprobed for tubulin and myc to confirm equal loading and similar expression of myc-tagged constructs respectively. To quantitate changes in Rev protein expression, blots were scanned and analyzed in Imagequant. Summary of 5 independent determinations is shown (D). (E) Effect of hnRNP E proteins on SEAP expression. Media from cells transfected with a SEAP expressing plasmid was harvested 48 hrs post transfection and SEAP expression normalized to that seen upon cotransfection with the CMVmyc control vector. Results shown are an average of 3 experiments. Error bars show standard deviation. SEAP expression of the myc control vector was set to 1.
    Pvdf Membrane, supplied by Beyotime, used in various techniques. Bioz Stars score: 99/100, based on 753 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore polyvinylidene difluoride pvdf
    hnRNP E1 but not hnRNP E2 inhibit expression of subgenomic HIV-1 expression vectors . (A) Schematic of HIV-1 env expression vector, pgTat, including the location of the ESE and ESS. The sequences of pgTat derivatives pgTatΔESE and pgTatΔESEΔESS SL1 are shown. (B) HeLa cells were co-transfected with pgTat or derivatives thereof, SVH6Rev, and hnRNP E expressing plasmids as indicated. 48 hours post-transfection, cells were harvested in RIPA buffer, and lysates fractionated on SDS-PAGE gels. Following transfer to <t>PVDF</t> membranes, blots were probed with antibody against gp120 and tubulin. (C) Effect of hnRNP E proteins on Gag/RRE expression. Shown is a diagram of the Gag expression construct used. HeLa cells were transfected with Gag/RRE, hnRNP E and Rev expression constructs as indicated. 48 hrs post transfection, cells were harvested, and lysates fractionated on SDS-PAGE gels. Blots were probed with antibody against Rev and <t>p24,</t> stripped then reprobed for tubulin and myc to confirm equal loading and similar expression of myc-tagged constructs respectively. To quantitate changes in Rev protein expression, blots were scanned and analyzed in Imagequant. Summary of 5 independent determinations is shown (D). (E) Effect of hnRNP E proteins on SEAP expression. Media from cells transfected with a SEAP expressing plasmid was harvested 48 hrs post transfection and SEAP expression normalized to that seen upon cotransfection with the CMVmyc control vector. Results shown are an average of 3 experiments. Error bars show standard deviation. SEAP expression of the myc control vector was set to 1.
    Polyvinylidene Difluoride Pvdf, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 405 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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 membranes
    <t>Bak</t> activation in HeLa cell mitochondria is temperature-dependent and influenced by Bak levels and soluble factors. A , mitochondria ( Mito. ) were freshly isolated from HeLa cells and incubated with 35 S-Bak at 37 or 22 °C for varying times. Following import, mitochondria were re-isolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE ( upper panel ) or resuspended in Laemmli sample buffer and subjected to SDS-PAGE ( lower panel ). Gels were visualized by autoradiography. B , isolated HeLa cell mitochondria in import buffer were either left alone or supplemented with rabbit reticulocyte lysate or 35 S-Bak containing rabbit reticulocyte lysate and incubated at 37 °C for the indicated times. Samples were solubilized in digitonin-containing buffer, separated by BN-PAGE and transferred to <t>PVDF</t> membrane. Following digital autoradiography ( left panel ), immunoblotting with antibodies against Bak was performed ( right panel ). C , experiment was performed as described in B ; however, the samples were separated by SDS-PAGE and subsequently transferred to PVDF. Following digital autoradiography ( upper panel ), immunoblotting was performed with antibodies against cytochrome c ( cyt. c, middle panel ) and B17.2L ( lower panel ).
    Pvdf Membranes, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 741 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Protein modification by PGA 1 -biotin and [1- 14 C]OPDA. ( A ) Proteins were extracted from leaves and incubated with (+) or without (-) PGA 1 -biotin [110 μM]. Protein samples (120 μg) were separated on a 10% SDS-PAGE and transferred to a PVDF membrane. Biotinylated proteins were detected by NeutrAvidin-HRP (left panel) followed by protein staining with Coomassie (loading control, right panel). ( B ) Leaf discs were exposed to [1- 14 C]OPDA for 2 h. Crude extract was centrifuged and an aliquot of the supernatant was analyzed by SDS-PAGE followed by radiography.

    Journal: PLoS ONE

    Article Title: TGA2 signaling in response to reactive electrophile species is not dependent on cysteine modification of TGA2

    doi: 10.1371/journal.pone.0195398

    Figure Lengend Snippet: Protein modification by PGA 1 -biotin and [1- 14 C]OPDA. ( A ) Proteins were extracted from leaves and incubated with (+) or without (-) PGA 1 -biotin [110 μM]. Protein samples (120 μg) were separated on a 10% SDS-PAGE and transferred to a PVDF membrane. Biotinylated proteins were detected by NeutrAvidin-HRP (left panel) followed by protein staining with Coomassie (loading control, right panel). ( B ) Leaf discs were exposed to [1- 14 C]OPDA for 2 h. Crude extract was centrifuged and an aliquot of the supernatant was analyzed by SDS-PAGE followed by radiography.

    Article Snippet: After SDS-PAGE, proteins were transferred to PVDF membrane (Merck, Darmstadt, Germany) and PGA1 -biotin labeled proteins were incubated with NeutrAvidin-HRP (Thermo Scientific, Bonn, Germany) at a dilution of 1:10,000 in TBST including 3% non-fat dried milk powder.

    Techniques: Modification, Incubation, SDS Page, Staining

    TGA2 modification by PGA 1 -biotin in vitro . Recombinant His-tagged TGA2 was incubated with 75 μM PGA 1 -biotin for 2 h. Proteins were separated on SDS-PAGE and transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP. (A) Stability of modification under different pH conditions tested at ambient temperature. (B) Incubation of TGA2 with PGA 1 -biotin in sodium phosphate, pH 7.5 at different temperatures. His-TGA2 has a predicted molecular weight of 42.3 kDa. The addition of six histidine residues may alter the migration slightly, resulting in a larger than expected protein band of ~50 kDa (Figs 2 and 3 ). Modification by PGA 1 -biotin would only add 0.6 kDa if a single site was modified. Immunoblot analysis with an αTGA2 antibody [ 22 ] confirmed the identity of His-TGA2 at 50 kDa (data not shown).

    Journal: PLoS ONE

    Article Title: TGA2 signaling in response to reactive electrophile species is not dependent on cysteine modification of TGA2

    doi: 10.1371/journal.pone.0195398

    Figure Lengend Snippet: TGA2 modification by PGA 1 -biotin in vitro . Recombinant His-tagged TGA2 was incubated with 75 μM PGA 1 -biotin for 2 h. Proteins were separated on SDS-PAGE and transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP. (A) Stability of modification under different pH conditions tested at ambient temperature. (B) Incubation of TGA2 with PGA 1 -biotin in sodium phosphate, pH 7.5 at different temperatures. His-TGA2 has a predicted molecular weight of 42.3 kDa. The addition of six histidine residues may alter the migration slightly, resulting in a larger than expected protein band of ~50 kDa (Figs 2 and 3 ). Modification by PGA 1 -biotin would only add 0.6 kDa if a single site was modified. Immunoblot analysis with an αTGA2 antibody [ 22 ] confirmed the identity of His-TGA2 at 50 kDa (data not shown).

    Article Snippet: After SDS-PAGE, proteins were transferred to PVDF membrane (Merck, Darmstadt, Germany) and PGA1 -biotin labeled proteins were incubated with NeutrAvidin-HRP (Thermo Scientific, Bonn, Germany) at a dilution of 1:10,000 in TBST including 3% non-fat dried milk powder.

    Techniques: Modification, In Vitro, Recombinant, Incubation, SDS Page, Molecular Weight, Migration

    Effect of cysteine blocking reagents on TGA2 modification by PGA 1 -biotin. Recombinant TGA2 was incubated with the cysteine reactive reagents N-ethylmaleimide (NEM), iodoacetamide (IAM) or S-methyl methanethiosulfonate (MMTS) prior to PGA 1 -biotin modification. Protein samples (30 μg) were separated on SDS-PAGE and transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP, followed by protein staining with Coomassie (loading control, lower panel).

    Journal: PLoS ONE

    Article Title: TGA2 signaling in response to reactive electrophile species is not dependent on cysteine modification of TGA2

    doi: 10.1371/journal.pone.0195398

    Figure Lengend Snippet: Effect of cysteine blocking reagents on TGA2 modification by PGA 1 -biotin. Recombinant TGA2 was incubated with the cysteine reactive reagents N-ethylmaleimide (NEM), iodoacetamide (IAM) or S-methyl methanethiosulfonate (MMTS) prior to PGA 1 -biotin modification. Protein samples (30 μg) were separated on SDS-PAGE and transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP, followed by protein staining with Coomassie (loading control, lower panel).

    Article Snippet: After SDS-PAGE, proteins were transferred to PVDF membrane (Merck, Darmstadt, Germany) and PGA1 -biotin labeled proteins were incubated with NeutrAvidin-HRP (Thermo Scientific, Bonn, Germany) at a dilution of 1:10,000 in TBST including 3% non-fat dried milk powder.

    Techniques: Blocking Assay, Modification, Recombinant, Incubation, SDS Page, Staining

    Cysteine specific modification of proteins by RES. Proteins were extracted from leaves and incubated without (control, Con) or with the cysteine reactive reagents N-ethylmaleimide (NEM), iodoacetamide (IAM), S-methyl methanethiosulfonate (MMTS) for 1 h prior to RES lipid modification. Protein samples were separated by SDS-PAGE. ( A ) After modification with PGA 1 -biotin and SDS-PAGE, proteins (90 μg) were transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP (upper panel) followed by protein staining using Coomassie blue (loading control, bottom panel). ( B ) After modification by [1- 14 C]OPDA (500000 cpm) for 4 h, the SDS–PAGE gel (loaded with 25 μg protein per lane) was analyzed by autoradiography (left panel) and Coomassie staining (right panel).

    Journal: PLoS ONE

    Article Title: TGA2 signaling in response to reactive electrophile species is not dependent on cysteine modification of TGA2

    doi: 10.1371/journal.pone.0195398

    Figure Lengend Snippet: Cysteine specific modification of proteins by RES. Proteins were extracted from leaves and incubated without (control, Con) or with the cysteine reactive reagents N-ethylmaleimide (NEM), iodoacetamide (IAM), S-methyl methanethiosulfonate (MMTS) for 1 h prior to RES lipid modification. Protein samples were separated by SDS-PAGE. ( A ) After modification with PGA 1 -biotin and SDS-PAGE, proteins (90 μg) were transferred to a PVDF membrane. Biotinylated proteins were visualized by NeutrAvidin-HRP (upper panel) followed by protein staining using Coomassie blue (loading control, bottom panel). ( B ) After modification by [1- 14 C]OPDA (500000 cpm) for 4 h, the SDS–PAGE gel (loaded with 25 μg protein per lane) was analyzed by autoradiography (left panel) and Coomassie staining (right panel).

    Article Snippet: After SDS-PAGE, proteins were transferred to PVDF membrane (Merck, Darmstadt, Germany) and PGA1 -biotin labeled proteins were incubated with NeutrAvidin-HRP (Thermo Scientific, Bonn, Germany) at a dilution of 1:10,000 in TBST including 3% non-fat dried milk powder.

    Techniques: Modification, Incubation, SDS Page, Staining, Autoradiography

    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

    SerpinB5 phosphorylation in the lactating mammary gland. A. 100 ug of protein extracts from mammary gland isolated on days 1, 10 and 20 of lactation (1L, 10L and 20L), and on days 1 and 5 of involution (1I and 5I) were resolved by 8% Phos-Tag™ SDS-PAGE gel. Proteins were transferred to PVDF membrane and probed with rabbit anti-SerpinB5 (Sigma). Arrows indicate two phosphorylated SerpinB5 bands (P- SerpinB5-1 and P- SerpinB5-2) and arrowhead indicates unphosphorylated SerpinB5. Top and middle panels represent a short and a long X-ray film exposure, respectively. The membrane was reincubated with anti-HSP90 for a loading control; B. each of the three bands was quantified and expressed as percentage of the total levels of detected SerpinB5. This result is representative of two independent experiments; C. 2 mg of protein extracts of mammary gland isolated on days 10 and 20 of lactation were analyzed by 2D-SDS-PAGE using 17 cm pH 4.7–5.9 IPG strips followed by Western blot with anti-SerpinB5 (Santa Cruz). This result is representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    doi: 10.1371/journal.pone.0159856

    Figure Lengend Snippet: SerpinB5 phosphorylation in the lactating mammary gland. A. 100 ug of protein extracts from mammary gland isolated on days 1, 10 and 20 of lactation (1L, 10L and 20L), and on days 1 and 5 of involution (1I and 5I) were resolved by 8% Phos-Tag™ SDS-PAGE gel. Proteins were transferred to PVDF membrane and probed with rabbit anti-SerpinB5 (Sigma). Arrows indicate two phosphorylated SerpinB5 bands (P- SerpinB5-1 and P- SerpinB5-2) and arrowhead indicates unphosphorylated SerpinB5. Top and middle panels represent a short and a long X-ray film exposure, respectively. The membrane was reincubated with anti-HSP90 for a loading control; B. each of the three bands was quantified and expressed as percentage of the total levels of detected SerpinB5. This result is representative of two independent experiments; C. 2 mg of protein extracts of mammary gland isolated on days 10 and 20 of lactation were analyzed by 2D-SDS-PAGE using 17 cm pH 4.7–5.9 IPG strips followed by Western blot with anti-SerpinB5 (Santa Cruz). This result is representative of two independent experiments.

    Article Snippet: 50 ug of nuclear (nuc) and cytoplasmic (cyt) protein fractions of a 1 day ( A ) or 5 day ( B ) involuting mammary gland were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Santa Cruz).

    Techniques: Isolation, SDS Page, Western Blot

    SerpinB5 subcellular localization in the mouse mammary gland. A. SerpinB5 immunofluorescence staining (Sigma) on paraffin section of a 10 day lactating gland; B. SerpinB5 and DAPI merge image; C. Representative area enlarged from boxed region indicating a SerpinB5-positive nucleus (white arrowhead), a partially-positive nucleus (yellow arrowhead) and a SerpinB5-negative nucleus (white arrow). D. 50 ug of nuclear (nuc) and cytoplasmic (cyt) protein fractions of a 10 day lactating mammary gland were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Santa Cruz). Fractionation efficiency was monitored by reprobing the membrane with anti-Lamin B1 and anti-alpha-tubulin. Scale bar—20 μm.

    Journal: PLoS ONE

    Article Title: EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    doi: 10.1371/journal.pone.0159856

    Figure Lengend Snippet: SerpinB5 subcellular localization in the mouse mammary gland. A. SerpinB5 immunofluorescence staining (Sigma) on paraffin section of a 10 day lactating gland; B. SerpinB5 and DAPI merge image; C. Representative area enlarged from boxed region indicating a SerpinB5-positive nucleus (white arrowhead), a partially-positive nucleus (yellow arrowhead) and a SerpinB5-negative nucleus (white arrow). D. 50 ug of nuclear (nuc) and cytoplasmic (cyt) protein fractions of a 10 day lactating mammary gland were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Santa Cruz). Fractionation efficiency was monitored by reprobing the membrane with anti-Lamin B1 and anti-alpha-tubulin. Scale bar—20 μm.

    Article Snippet: 50 ug of nuclear (nuc) and cytoplasmic (cyt) protein fractions of a 1 day ( A ) or 5 day ( B ) involuting mammary gland were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Santa Cruz).

    Techniques: Immunofluorescence, Staining, Paraffin Section, SDS Page, Fractionation

    SerpinB5 expression is developmentally regulated in the mouse mammary gland. A. 100 μg of protein extracts from mammary glands isolated on days 3, 10, 18 of pregnancy (3P, 10P and 18P), on days 1, 10 and 20 of lactation (1L, 10L and 20L), on days 1 and 5 of involution (1I and 5I) and from virgin mice (V), were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with mouse monoclonal anti-SerpinB5 (Millipore). 50 ug of MCF-10A protein extract was loaded as a positive control (lane 10). The membrane was reprobed with anti-alpha-tubulin for loading control (lower panel); B. The bar graph shows SerpinB5 levels normalized by alpha-tubulin. This result is representative of 3 independent experiments done with two different mouse strains.

    Journal: PLoS ONE

    Article Title: EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    doi: 10.1371/journal.pone.0159856

    Figure Lengend Snippet: SerpinB5 expression is developmentally regulated in the mouse mammary gland. A. 100 μg of protein extracts from mammary glands isolated on days 3, 10, 18 of pregnancy (3P, 10P and 18P), on days 1, 10 and 20 of lactation (1L, 10L and 20L), on days 1 and 5 of involution (1I and 5I) and from virgin mice (V), were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with mouse monoclonal anti-SerpinB5 (Millipore). 50 ug of MCF-10A protein extract was loaded as a positive control (lane 10). The membrane was reprobed with anti-alpha-tubulin for loading control (lower panel); B. The bar graph shows SerpinB5 levels normalized by alpha-tubulin. This result is representative of 3 independent experiments done with two different mouse strains.

    Article Snippet: 50 ug of nuclear (nuc) and cytoplasmic (cyt) protein fractions of a 1 day ( A ) or 5 day ( B ) involuting mammary gland were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Santa Cruz).

    Techniques: Expressing, Isolation, Mouse Assay, SDS Page, Positive Control

    EGF and TGF alpha regulate SerpinB5 phosphorylation in MCF-10A cells. A . Starved MCF-10A cells were left untreated (upper panel) or treated with 20 ng/ml with EGF (middle panel) or TGF alpha (lower panel) for 20 minutes. 1.7 mg of protein extracts were analyzed by 2D-SDS-PAGE using 17 cm pH 4.7–5.9 IPG strips followed by Western blot with anti-SerpinB5 (BD Pharmigen); B. Starved MCF-10A cells were left untreated (lane 1) or treated with 20 ng/ml of EGF for the indicated periods of time (lanes 2–4). 50 ug of whole protein extracts were resolved by 8% Phos-Tag™ SDS-PAGE gel to separate the phosphorylated species. Proteins were transferred to PVDF membrane and probed with rabbit anti-SerpinB5 (Santa Cruz); C. MCF-10A cells were grown in complete medium (lane 1) or starved medium without (lane 2) or with 20 ng/ml of EGF for 15 min (lane 3). 50 ug of whole protein extracts were resolved by 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Millipore) or anti-alpha-tubulin for a loading control. This result is representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    doi: 10.1371/journal.pone.0159856

    Figure Lengend Snippet: EGF and TGF alpha regulate SerpinB5 phosphorylation in MCF-10A cells. A . Starved MCF-10A cells were left untreated (upper panel) or treated with 20 ng/ml with EGF (middle panel) or TGF alpha (lower panel) for 20 minutes. 1.7 mg of protein extracts were analyzed by 2D-SDS-PAGE using 17 cm pH 4.7–5.9 IPG strips followed by Western blot with anti-SerpinB5 (BD Pharmigen); B. Starved MCF-10A cells were left untreated (lane 1) or treated with 20 ng/ml of EGF for the indicated periods of time (lanes 2–4). 50 ug of whole protein extracts were resolved by 8% Phos-Tag™ SDS-PAGE gel to separate the phosphorylated species. Proteins were transferred to PVDF membrane and probed with rabbit anti-SerpinB5 (Santa Cruz); C. MCF-10A cells were grown in complete medium (lane 1) or starved medium without (lane 2) or with 20 ng/ml of EGF for 15 min (lane 3). 50 ug of whole protein extracts were resolved by 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Millipore) or anti-alpha-tubulin for a loading control. This result is representative of two independent experiments.

    Article Snippet: 50 ug of nuclear (nuc) and cytoplasmic (cyt) protein fractions of a 1 day ( A ) or 5 day ( B ) involuting mammary gland were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Santa Cruz).

    Techniques: SDS Page, Western Blot

    AREG is responsible for SerpinB5 phosphorylation in starved-MCF-10A cells. Starved MCF-10A cells (lane 1) were grown in medium containing either 1 ug/ml of goat IgG (lane 2) or goat anti-AREG-neutralizing antibody (lane 3) for 24 h. 50 ug whole protein extracts were resolved by 8% Phos-Tag™ SDS-PAGE gel. Recombinant SerpinB5 was used as unphosphorylated control (lane 4). Proteins were transferred to PVDF membrane and probed with anti-SerpinB5 (Sigma). A short exposure is shown on the right side; Asterisk indicates a spurious band. This result is representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    doi: 10.1371/journal.pone.0159856

    Figure Lengend Snippet: AREG is responsible for SerpinB5 phosphorylation in starved-MCF-10A cells. Starved MCF-10A cells (lane 1) were grown in medium containing either 1 ug/ml of goat IgG (lane 2) or goat anti-AREG-neutralizing antibody (lane 3) for 24 h. 50 ug whole protein extracts were resolved by 8% Phos-Tag™ SDS-PAGE gel. Recombinant SerpinB5 was used as unphosphorylated control (lane 4). Proteins were transferred to PVDF membrane and probed with anti-SerpinB5 (Sigma). A short exposure is shown on the right side; Asterisk indicates a spurious band. This result is representative of two independent experiments.

    Article Snippet: 50 ug of nuclear (nuc) and cytoplasmic (cyt) protein fractions of a 1 day ( A ) or 5 day ( B ) involuting mammary gland were subjected to 12% SDS-PAGE, transferred to PVDF membrane and probed with anti-SerpinB5 (Santa Cruz).

    Techniques: SDS Page, Recombinant

    Derivatives of Yersinia pestis KIM8-3002 (KIM5 pPCP1-minus, Sm r ) were grown in a chemically defined medium at 26°C for 2 h in the presence (lanes 1, 3, and 5) or absence of calcium (lanes 2, 4, and 6). Lanes 1 and 2 contain Y. pestis KIM8-3002. Lanes 3 and 4 contain Y. pestis KIM8-3002 Δ yscF expressing YscF from pBAD18-YscF. Lanes 5 and 6 contain Y. pestis KIM8-3002 Δ yscF gene. After the 2 h growth, the culture was shifted to 37°C to induce expression of the Ysc type III secretion system and the Low Calcium Response. Following 4 h of growth at 37°C cultures, were centrifuged to obtain whole cell fractions and cell-free culture supernatant fractions. Total proteins from each fraction were precipitated with 10% trichloro acetic acid. Dried proteins were resuspended in SDS-PAGE sample buffer and electrophoresed in a 15% SDS-PAGE gel. Proteins were then transferred to a PVDF membrane and immuno-blotted with pooled mouse serum used at a 1:20,000 dilution. Mouse serum was obtained by bleeding mice subsequent to immunization with HT-YscF, serum from several mice was pooled to control for animal specific variation. The position and sizes for the molecular weight markers are indicated and the position of YscF is shown.

    Journal: BMC Microbiology

    Article Title: Immunization of mice with YscF provides protection from Yersinia pestis infections

    doi: 10.1186/1471-2180-5-38

    Figure Lengend Snippet: Derivatives of Yersinia pestis KIM8-3002 (KIM5 pPCP1-minus, Sm r ) were grown in a chemically defined medium at 26°C for 2 h in the presence (lanes 1, 3, and 5) or absence of calcium (lanes 2, 4, and 6). Lanes 1 and 2 contain Y. pestis KIM8-3002. Lanes 3 and 4 contain Y. pestis KIM8-3002 Δ yscF expressing YscF from pBAD18-YscF. Lanes 5 and 6 contain Y. pestis KIM8-3002 Δ yscF gene. After the 2 h growth, the culture was shifted to 37°C to induce expression of the Ysc type III secretion system and the Low Calcium Response. Following 4 h of growth at 37°C cultures, were centrifuged to obtain whole cell fractions and cell-free culture supernatant fractions. Total proteins from each fraction were precipitated with 10% trichloro acetic acid. Dried proteins were resuspended in SDS-PAGE sample buffer and electrophoresed in a 15% SDS-PAGE gel. Proteins were then transferred to a PVDF membrane and immuno-blotted with pooled mouse serum used at a 1:20,000 dilution. Mouse serum was obtained by bleeding mice subsequent to immunization with HT-YscF, serum from several mice was pooled to control for animal specific variation. The position and sizes for the molecular weight markers are indicated and the position of YscF is shown.

    Article Snippet: Samples were boiled for 5 min and loaded onto 12.5 % SDS-PAGE gels, immunoblotted to PVDF membranes and probed with Elk-1 (#9182) or phosphospecific Elk-1 (#9181) antibody preparations (Cell Signaling Technology).

    Techniques: Expressing, SDS Page, Mouse Assay, Molecular Weight

    Purified HT-YscF was run on SDS-PAGE gels (12.5% for Panels A and B, 15% Panel C) to analyze purity of the recovered protein. Panel A, separated proteins were stained with GelCode Blue stain (Pierce Chemical). Panel B, purified HT-YscF was immunoblotted to PVDF and probed with antibody specific for penta-histidine to identify which bands contained the His-tag. Panel C, purified HT-YscF was immunoblotted to PVDF and probed with post-immune serum from immunized mice.

    Journal: BMC Microbiology

    Article Title: Immunization of mice with YscF provides protection from Yersinia pestis infections

    doi: 10.1186/1471-2180-5-38

    Figure Lengend Snippet: Purified HT-YscF was run on SDS-PAGE gels (12.5% for Panels A and B, 15% Panel C) to analyze purity of the recovered protein. Panel A, separated proteins were stained with GelCode Blue stain (Pierce Chemical). Panel B, purified HT-YscF was immunoblotted to PVDF and probed with antibody specific for penta-histidine to identify which bands contained the His-tag. Panel C, purified HT-YscF was immunoblotted to PVDF and probed with post-immune serum from immunized mice.

    Article Snippet: Samples were boiled for 5 min and loaded onto 12.5 % SDS-PAGE gels, immunoblotted to PVDF membranes and probed with Elk-1 (#9182) or phosphospecific Elk-1 (#9181) antibody preparations (Cell Signaling Technology).

    Techniques: Purification, SDS Page, Staining, Mouse Assay

    Y. pestis strain KIM8-3002 (wt) (lane 1 and lanes 3–6) and KIM8-3002.2 (Δ yopD ) (lane 2 and lanes 7–10) both containing plasmid pYopE 129 -Elk were used to infect HeLa cells at an MOI of 10. 4 h following infection the culture supernatant containing non-adherent bacteria were removed the remaining adherent HeLa cells were solubilized in 2X SDS-PAGE buffer. Following solubilization proteins were separated and immunoblotted to triplicate PVDF membranes. The triplicate blots were probed with α-YopE, α-Elk, or α-PO 4 -Elk, followed by incubation with an alkaline phosphatase conjugated secondary antibody and developed using NBT/BCIP. To some samples anti-sera specific for HT-YscF (lanes 3–4 and lanes 7–8) or the Pseudomonas protein, PcrG (lanes 5–6 and lanes 9–10), were added at dilutions of 1:10 (lanes 3, 5, 7, and 9) or 1:25 (lanes 4, 6, 8, and 10).

    Journal: BMC Microbiology

    Article Title: Immunization of mice with YscF provides protection from Yersinia pestis infections

    doi: 10.1186/1471-2180-5-38

    Figure Lengend Snippet: Y. pestis strain KIM8-3002 (wt) (lane 1 and lanes 3–6) and KIM8-3002.2 (Δ yopD ) (lane 2 and lanes 7–10) both containing plasmid pYopE 129 -Elk were used to infect HeLa cells at an MOI of 10. 4 h following infection the culture supernatant containing non-adherent bacteria were removed the remaining adherent HeLa cells were solubilized in 2X SDS-PAGE buffer. Following solubilization proteins were separated and immunoblotted to triplicate PVDF membranes. The triplicate blots were probed with α-YopE, α-Elk, or α-PO 4 -Elk, followed by incubation with an alkaline phosphatase conjugated secondary antibody and developed using NBT/BCIP. To some samples anti-sera specific for HT-YscF (lanes 3–4 and lanes 7–8) or the Pseudomonas protein, PcrG (lanes 5–6 and lanes 9–10), were added at dilutions of 1:10 (lanes 3, 5, 7, and 9) or 1:25 (lanes 4, 6, 8, and 10).

    Article Snippet: Samples were boiled for 5 min and loaded onto 12.5 % SDS-PAGE gels, immunoblotted to PVDF membranes and probed with Elk-1 (#9182) or phosphospecific Elk-1 (#9181) antibody preparations (Cell Signaling Technology).

    Techniques: Plasmid Preparation, Infection, SDS Page, Incubation

    hnRNP E1 but not hnRNP E2 inhibit expression of subgenomic HIV-1 expression vectors . (A) Schematic of HIV-1 env expression vector, pgTat, including the location of the ESE and ESS. The sequences of pgTat derivatives pgTatΔESE and pgTatΔESEΔESS SL1 are shown. (B) HeLa cells were co-transfected with pgTat or derivatives thereof, SVH6Rev, and hnRNP E expressing plasmids as indicated. 48 hours post-transfection, cells were harvested in RIPA buffer, and lysates fractionated on SDS-PAGE gels. Following transfer to PVDF membranes, blots were probed with antibody against gp120 and tubulin. (C) Effect of hnRNP E proteins on Gag/RRE expression. Shown is a diagram of the Gag expression construct used. HeLa cells were transfected with Gag/RRE, hnRNP E and Rev expression constructs as indicated. 48 hrs post transfection, cells were harvested, and lysates fractionated on SDS-PAGE gels. Blots were probed with antibody against Rev and p24, stripped then reprobed for tubulin and myc to confirm equal loading and similar expression of myc-tagged constructs respectively. To quantitate changes in Rev protein expression, blots were scanned and analyzed in Imagequant. Summary of 5 independent determinations is shown (D). (E) Effect of hnRNP E proteins on SEAP expression. Media from cells transfected with a SEAP expressing plasmid was harvested 48 hrs post transfection and SEAP expression normalized to that seen upon cotransfection with the CMVmyc control vector. Results shown are an average of 3 experiments. Error bars show standard deviation. SEAP expression of the myc control vector was set to 1.

    Journal: Retrovirology

    Article Title: hnRNP E1 and E2 have distinct roles in modulating HIV-1 gene expression

    doi: 10.1186/1742-4690-4-28

    Figure Lengend Snippet: hnRNP E1 but not hnRNP E2 inhibit expression of subgenomic HIV-1 expression vectors . (A) Schematic of HIV-1 env expression vector, pgTat, including the location of the ESE and ESS. The sequences of pgTat derivatives pgTatΔESE and pgTatΔESEΔESS SL1 are shown. (B) HeLa cells were co-transfected with pgTat or derivatives thereof, SVH6Rev, and hnRNP E expressing plasmids as indicated. 48 hours post-transfection, cells were harvested in RIPA buffer, and lysates fractionated on SDS-PAGE gels. Following transfer to PVDF membranes, blots were probed with antibody against gp120 and tubulin. (C) Effect of hnRNP E proteins on Gag/RRE expression. Shown is a diagram of the Gag expression construct used. HeLa cells were transfected with Gag/RRE, hnRNP E and Rev expression constructs as indicated. 48 hrs post transfection, cells were harvested, and lysates fractionated on SDS-PAGE gels. Blots were probed with antibody against Rev and p24, stripped then reprobed for tubulin and myc to confirm equal loading and similar expression of myc-tagged constructs respectively. To quantitate changes in Rev protein expression, blots were scanned and analyzed in Imagequant. Summary of 5 independent determinations is shown (D). (E) Effect of hnRNP E proteins on SEAP expression. Media from cells transfected with a SEAP expressing plasmid was harvested 48 hrs post transfection and SEAP expression normalized to that seen upon cotransfection with the CMVmyc control vector. Results shown are an average of 3 experiments. Error bars show standard deviation. SEAP expression of the myc control vector was set to 1.

    Article Snippet: Western blots Proteins were fractionated on sodium dodecyl sulfate-7% (gp160/120) or 10% polyacrylamide gels (Rev, p24, hnRNP E1, hnRNP E2, myc and tubulin) and transferred to PVDF membrane (PALL Corporation).

    Techniques: Expressing, Plasmid Preparation, Transfection, SDS Page, Construct, Cotransfection, Standard Deviation

    hnRNP E1 but not E2 overexpression decreases HIV-1 structural protein levels . 293 cells were transfected with mycE1, mycE2 or the empty CMVmyc vector, alongside provirus HxBruR-/RI- andCMVPLAP. 48 hrs post transfection, cells were lifted in 1×PBS and a fraction (1/4) of the cells lysed in 9 M urea, 5 mM Tris pH8 and fractionated on SDS-PAGE gels. Protein was transferred to PVDF membrane and probed with antibody to p24. Blots were stripped in 62.5 mM TrisHCl pH 6.7, 2%SDS, 100 mM β-mercaptoethanol and reprobed with antibodies to gp160/120 (α gp120), hnRNP E1 and E2, myc and tubulin as indicated. (A) Results of western blotting. Antibodies and transfected plasmids are as indicated. (B) Relative SEAP expression in transfected cells. Levels were normalized to that of the empty CMVmyc vector control. Results shown are an average of three experiments. Error bars represent standard deviation.

    Journal: Retrovirology

    Article Title: hnRNP E1 and E2 have distinct roles in modulating HIV-1 gene expression

    doi: 10.1186/1742-4690-4-28

    Figure Lengend Snippet: hnRNP E1 but not E2 overexpression decreases HIV-1 structural protein levels . 293 cells were transfected with mycE1, mycE2 or the empty CMVmyc vector, alongside provirus HxBruR-/RI- andCMVPLAP. 48 hrs post transfection, cells were lifted in 1×PBS and a fraction (1/4) of the cells lysed in 9 M urea, 5 mM Tris pH8 and fractionated on SDS-PAGE gels. Protein was transferred to PVDF membrane and probed with antibody to p24. Blots were stripped in 62.5 mM TrisHCl pH 6.7, 2%SDS, 100 mM β-mercaptoethanol and reprobed with antibodies to gp160/120 (α gp120), hnRNP E1 and E2, myc and tubulin as indicated. (A) Results of western blotting. Antibodies and transfected plasmids are as indicated. (B) Relative SEAP expression in transfected cells. Levels were normalized to that of the empty CMVmyc vector control. Results shown are an average of three experiments. Error bars represent standard deviation.

    Article Snippet: Western blots Proteins were fractionated on sodium dodecyl sulfate-7% (gp160/120) or 10% polyacrylamide gels (Rev, p24, hnRNP E1, hnRNP E2, myc and tubulin) and transferred to PVDF membrane (PALL Corporation).

    Techniques: Over Expression, Transfection, Plasmid Preparation, SDS Page, Western Blot, Expressing, Standard Deviation

    Depletion of hnRNP E1 and E2 alters HIV-1 gene expression . 293 cells were transfected with siRNAs to hnRNP E1(E1(3) or E1(16)), hnRNP E2 (E2) or a scrambled siRNA control (scr)using Oligofectamine. 24 hrs later, cells were transfected withHxBruR-/RI- provirus and CMVPLAP using Fugene. 48 hrs post transfection, cells were harvested, lysates fractionated on SDS-PAGE gels and analyzed by western blot. (A) Results of western blotting. Proteins examined and siRNAs used are as indicated. (B) Kinetics of siRNA depletion of hnRNP E1/E2. Cells were treated with control siRNA (scrambled) or directed to hnRNP E1 (E1(3), E1(16)), or hnRNP E2 (E2) and harvested 24 h after transfection. Cell extracts were fractionated on SDS PAGE gels, transferred to PVDF membrane and probed with antibody to hnRNP E1 (E1), hnRNP E2 (E2), or tubulin (tubulin). (C) Relative SEAP expression in transfected cells. Media was harvested 48 hrs post transfection and assayed for SEAP activity. Levels were normalized to that of the scrambled siRNA control. Results shown are an average of three experiments. Error bars represent standard deviation. (D) Northern analysis of the effect of hnRNP E1/E2 depletion on HIV RNA levels. Cells were treated with siRNAs as above and total RNA extracted. Following fractionation on formaldehyde agarose gels and transfer to nitrocellulose membrane, blots were probed to detect viral RNAs (9, 4, and 2 kb). Blots were reprobed for GAPDH RNA to confirm equal loading. Quantitation of 3 experiments is shown at right. Results were normalized to GAPDH with the data for the scrambled siRNA sample (scr) set at 1. Error bars show standard deviation.

    Journal: Retrovirology

    Article Title: hnRNP E1 and E2 have distinct roles in modulating HIV-1 gene expression

    doi: 10.1186/1742-4690-4-28

    Figure Lengend Snippet: Depletion of hnRNP E1 and E2 alters HIV-1 gene expression . 293 cells were transfected with siRNAs to hnRNP E1(E1(3) or E1(16)), hnRNP E2 (E2) or a scrambled siRNA control (scr)using Oligofectamine. 24 hrs later, cells were transfected withHxBruR-/RI- provirus and CMVPLAP using Fugene. 48 hrs post transfection, cells were harvested, lysates fractionated on SDS-PAGE gels and analyzed by western blot. (A) Results of western blotting. Proteins examined and siRNAs used are as indicated. (B) Kinetics of siRNA depletion of hnRNP E1/E2. Cells were treated with control siRNA (scrambled) or directed to hnRNP E1 (E1(3), E1(16)), or hnRNP E2 (E2) and harvested 24 h after transfection. Cell extracts were fractionated on SDS PAGE gels, transferred to PVDF membrane and probed with antibody to hnRNP E1 (E1), hnRNP E2 (E2), or tubulin (tubulin). (C) Relative SEAP expression in transfected cells. Media was harvested 48 hrs post transfection and assayed for SEAP activity. Levels were normalized to that of the scrambled siRNA control. Results shown are an average of three experiments. Error bars represent standard deviation. (D) Northern analysis of the effect of hnRNP E1/E2 depletion on HIV RNA levels. Cells were treated with siRNAs as above and total RNA extracted. Following fractionation on formaldehyde agarose gels and transfer to nitrocellulose membrane, blots were probed to detect viral RNAs (9, 4, and 2 kb). Blots were reprobed for GAPDH RNA to confirm equal loading. Quantitation of 3 experiments is shown at right. Results were normalized to GAPDH with the data for the scrambled siRNA sample (scr) set at 1. Error bars show standard deviation.

    Article Snippet: Western blots Proteins were fractionated on sodium dodecyl sulfate-7% (gp160/120) or 10% polyacrylamide gels (Rev, p24, hnRNP E1, hnRNP E2, myc and tubulin) and transferred to PVDF membrane (PALL Corporation).

    Techniques: Expressing, Transfection, SDS Page, Western Blot, Activity Assay, Standard Deviation, Northern Blot, Fractionation, Quantitation Assay

    Suppression of HIV-1 gene expression by hnRNP E1 is dependent upon the C-terminal KH domain . (A) Schematic of hnRNP E1 and domain mutants thereof. Light grey boxes denote the KH domains. Dark shading denotes hnRNP E2 sequence and white indicates hnRNP E1 sequence. (B) Localization of hnRNP E1 and domain mutants and hnRNP E2. HeLa cells were grown on coverslips and transfected with the hnRNP E expressing plasmids. 48 hrs post transfection, cells were washed with 1× PBS, fixed in 4% paraformaldehyde, 1× PBS and localization of transfected hnRNP E proteins (anti-myc) and nuclei (DAPI) determined. Magnification is 630×. Shown are representative examples of results obtained in multiple trials. (C) 293T cells were transfected with mycE1/E2 expressing plasmids as indicated along with pgTat and SVH6Rev. 48hrs post transfection, cells were harvested and lysates fractionated on SDS-PAGE gels. Following transfer to PVDF membranes, blots were probed with antibody to gp120, tubulin, rev or the myc tag. Cell supernatants were analyzed for levels of SEAP expression (D).

    Journal: Retrovirology

    Article Title: hnRNP E1 and E2 have distinct roles in modulating HIV-1 gene expression

    doi: 10.1186/1742-4690-4-28

    Figure Lengend Snippet: Suppression of HIV-1 gene expression by hnRNP E1 is dependent upon the C-terminal KH domain . (A) Schematic of hnRNP E1 and domain mutants thereof. Light grey boxes denote the KH domains. Dark shading denotes hnRNP E2 sequence and white indicates hnRNP E1 sequence. (B) Localization of hnRNP E1 and domain mutants and hnRNP E2. HeLa cells were grown on coverslips and transfected with the hnRNP E expressing plasmids. 48 hrs post transfection, cells were washed with 1× PBS, fixed in 4% paraformaldehyde, 1× PBS and localization of transfected hnRNP E proteins (anti-myc) and nuclei (DAPI) determined. Magnification is 630×. Shown are representative examples of results obtained in multiple trials. (C) 293T cells were transfected with mycE1/E2 expressing plasmids as indicated along with pgTat and SVH6Rev. 48hrs post transfection, cells were harvested and lysates fractionated on SDS-PAGE gels. Following transfer to PVDF membranes, blots were probed with antibody to gp120, tubulin, rev or the myc tag. Cell supernatants were analyzed for levels of SEAP expression (D).

    Article Snippet: Western blots Proteins were fractionated on sodium dodecyl sulfate-7% (gp160/120) or 10% polyacrylamide gels (Rev, p24, hnRNP E1, hnRNP E2, myc and tubulin) and transferred to PVDF membrane (PALL Corporation).

    Techniques: Expressing, Sequencing, Transfection, 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

    Bak activation in HeLa cell mitochondria is temperature-dependent and influenced by Bak levels and soluble factors. A , mitochondria ( Mito. ) were freshly isolated from HeLa cells and incubated with 35 S-Bak at 37 or 22 °C for varying times. Following import, mitochondria were re-isolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE ( upper panel ) or resuspended in Laemmli sample buffer and subjected to SDS-PAGE ( lower panel ). Gels were visualized by autoradiography. B , isolated HeLa cell mitochondria in import buffer were either left alone or supplemented with rabbit reticulocyte lysate or 35 S-Bak containing rabbit reticulocyte lysate and incubated at 37 °C for the indicated times. Samples were solubilized in digitonin-containing buffer, separated by BN-PAGE and transferred to PVDF membrane. Following digital autoradiography ( left panel ), immunoblotting with antibodies against Bak was performed ( right panel ). C , experiment was performed as described in B ; however, the samples were separated by SDS-PAGE and subsequently transferred to PVDF. Following digital autoradiography ( upper panel ), immunoblotting was performed with antibodies against cytochrome c ( cyt. c, middle panel ) and B17.2L ( lower panel ).

    Journal: The Journal of Biological Chemistry

    Article Title: Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor *

    doi: 10.1074/jbc.M110.159301

    Figure Lengend Snippet: Bak activation in HeLa cell mitochondria is temperature-dependent and influenced by Bak levels and soluble factors. A , mitochondria ( Mito. ) were freshly isolated from HeLa cells and incubated with 35 S-Bak at 37 or 22 °C for varying times. Following import, mitochondria were re-isolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE ( upper panel ) or resuspended in Laemmli sample buffer and subjected to SDS-PAGE ( lower panel ). Gels were visualized by autoradiography. B , isolated HeLa cell mitochondria in import buffer were either left alone or supplemented with rabbit reticulocyte lysate or 35 S-Bak containing rabbit reticulocyte lysate and incubated at 37 °C for the indicated times. Samples were solubilized in digitonin-containing buffer, separated by BN-PAGE and transferred to PVDF membrane. Following digital autoradiography ( left panel ), immunoblotting with antibodies against Bak was performed ( right panel ). C , experiment was performed as described in B ; however, the samples were separated by SDS-PAGE and subsequently transferred to PVDF. Following digital autoradiography ( upper panel ), immunoblotting was performed with antibodies against cytochrome c ( cyt. c, middle panel ) and B17.2L ( lower panel ).

    Article Snippet: For Western blot analysis, gels were transferred to PVDF membranes and probed with antibodies against Bak 7D10 , Tom40 , B17.2L , Hsc70 , Tim23 (BD Biosciences), cytochrome c (BD Biosciences), and mtHsp70 (provided by Nick Hoogenraad, Melbourne, Australia).

    Techniques: Activation Assay, Isolation, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Autoradiography

    Release of Bak from a 400-kDa complex in isolated HeLa cell mitochondria induces MOMP. A , mitochondria were freshly isolated from HeLa cells and incubated with radiolabeled Bak precursor at 37 °C for varying times. Following import, mitochondria were reisolated and left untreated or treated with proteinase K. Mitochondria were then lysed in digitonin-containing buffer and subjected to BN-PAGE ( upper panel ) or resuspended in Laemmli sample buffer and subjected to SDS-PAGE ( lower panel ). Both gels were transferred to PVDF membrane prior to digital autoradiography. Two molecular weight assemblies of Bak are indicated (termed Bak I and Bak II ). Proteolytic fragments of Bak are indicated ( Bak *). # indicates nonspecific band. Lysate indicates a sample of the translated 35 S-Bak product. B , following autoradiography, the SDS-PAGE PVDF membrane from A was subjected to immunoblot analysis with antibodies against cytochrome c ( cyt. c ) and Tom40 (as control). 25 μg of total mitochondria protein (shown as mito in lane 11 ) was loaded as a control. C , 35 S-Bak was incubated with isolated HeLa cell mitochondria at 37 °C for 5 min. Mitochondria were isolated and resuspended in import buffer and then left untreated or treated with tBid and incubated for a further 10 min at 37 °C. Samples were split and analyzed by BN-PAGE ( upper panel ) and SDS-PAGE ( lower panel ) and then subjected to digital autoradiography. D , HeLa cell mitochondria treated with or without tBid were subjected to SDS-PAGE and probed with antibodies against cytochrome c and Tom40. E , isolated HeLa cell mitochondria were resuspended in import buffer with or without tBid for 10 min at 37 °C. Mitochondria were re-isolated, solubilized in digitonin-containing buffer, and separated by BN-PAGE followed by Western blot analysis using antibodies against Bak.

    Journal: The Journal of Biological Chemistry

    Article Title: Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor *

    doi: 10.1074/jbc.M110.159301

    Figure Lengend Snippet: Release of Bak from a 400-kDa complex in isolated HeLa cell mitochondria induces MOMP. A , mitochondria were freshly isolated from HeLa cells and incubated with radiolabeled Bak precursor at 37 °C for varying times. Following import, mitochondria were reisolated and left untreated or treated with proteinase K. Mitochondria were then lysed in digitonin-containing buffer and subjected to BN-PAGE ( upper panel ) or resuspended in Laemmli sample buffer and subjected to SDS-PAGE ( lower panel ). Both gels were transferred to PVDF membrane prior to digital autoradiography. Two molecular weight assemblies of Bak are indicated (termed Bak I and Bak II ). Proteolytic fragments of Bak are indicated ( Bak *). # indicates nonspecific band. Lysate indicates a sample of the translated 35 S-Bak product. B , following autoradiography, the SDS-PAGE PVDF membrane from A was subjected to immunoblot analysis with antibodies against cytochrome c ( cyt. c ) and Tom40 (as control). 25 μg of total mitochondria protein (shown as mito in lane 11 ) was loaded as a control. C , 35 S-Bak was incubated with isolated HeLa cell mitochondria at 37 °C for 5 min. Mitochondria were isolated and resuspended in import buffer and then left untreated or treated with tBid and incubated for a further 10 min at 37 °C. Samples were split and analyzed by BN-PAGE ( upper panel ) and SDS-PAGE ( lower panel ) and then subjected to digital autoradiography. D , HeLa cell mitochondria treated with or without tBid were subjected to SDS-PAGE and probed with antibodies against cytochrome c and Tom40. E , isolated HeLa cell mitochondria were resuspended in import buffer with or without tBid for 10 min at 37 °C. Mitochondria were re-isolated, solubilized in digitonin-containing buffer, and separated by BN-PAGE followed by Western blot analysis using antibodies against Bak.

    Article Snippet: For Western blot analysis, gels were transferred to PVDF membranes and probed with antibodies against Bak 7D10 , Tom40 , B17.2L , Hsc70 , Tim23 (BD Biosciences), cytochrome c (BD Biosciences), and mtHsp70 (provided by Nick Hoogenraad, Melbourne, Australia).

    Techniques: Isolation, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Autoradiography, Molecular Weight, Western Blot