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Roche na3 vo4
Na3 Vo4, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 489 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/na3 vo4/product/Roche
Average 94 stars, based on 489 article reviews
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na3 vo4 - by Bioz Stars, 2020-07
94/100 stars

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Protease Inhibitor:

Article Title: Reduced amyloidogenic processing of the amyloid ?-protein precursor by the small-molecule Differentiation Inducing Factor-1
Article Snippet: .. Cells were lysed in ice cold RIPA buffer (20 mM Tris–HCl (pH 7.4) containing 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (NP-40), 50 mM NaF, 1 mM Na3 VO4 , 1 mM Na2 MoO4 , 10 mg/mL aprotinin, and 10 mg/mL leupeptin), and supplemented with protease inhibitor tablets (Roche). .. Protein extracts were fractionated by SDS-PAGE and transferred to PVDF membranes.

Article Title: The Genetic and Biochemical Basis of FANCD2 Monoubiquitination
Article Snippet: .. In brief, to purify a FA core complex, ΔB/B-GS, or ΔB/B-GS/ΔC DT40 cells were lysed in GS buffer (50 mM HEPES [pH 8.0], 150 mM NaCl, 5% glycerol, 0.1% Igepal CA-630, 1.5 mM MgCl2 , 25 mM NaF, 2 mM Na3 VO4 , 40 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 mM β-mercaptoethanol [BME], and PhosSTOP inhibitor cocktail [Roche] and protease inhibitor cocktail) by passing through 19 G and 25 G needles multiple times. .. After clarification, the lysate was incubated with IgG-agarose beads (Sigma-Aldrich) for 2 hr with gentle rotation.

Article Title: Elevated Serum Melatonin under Constant Darkness Enhances Neural Repair in Spinal Cord Injury through Regulation of Circadian Clock Proteins Expression
Article Snippet: .. Protein Extraction and Western Blotting The spinal segments were lysed in buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM NaF, 1 mM Na3 VO4 , 1% NP-40, 0.25% sodium deoxycholate, and protease inhibitor cocktail; Roche diagnostics GmbH, Mannheim, Germany). ..

Article Title: Saponin monomer 13 of dwarf lilyturf tuber (DT-13) protects serum withdrawal-induced apoptosis through PI3K/Akt in HUVEC
Article Snippet: .. After treatments, cultured HUVEC were washed with cold PBS and lysed in a buffer containing 50 mM Tris–HCl, pH 7.4, 1% NP-40, 2% SDS, 0.1% deoxycholic acid, 0.1 mM EDTA, 0.1 mM EGTA, 2.5 mM sodium pyrophosphate, 5 mM sodium fluoride, 1 mM Na3- VO4 and protease inhibitor (Roche 11836145001). .. Proteins were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA).

Article Title: Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis
Article Snippet: .. Cell lysate was made in cell lysis buffer (20 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerol phosphate, 1 mmol/L Na3 VO4 ); and 1 mmol/L PMSF and protease inhibitor cocktail (04693116001, Roche) were added freshly. .. Protein concentration was determined by Bio-Rad Protein Assay Kit II (5000002, Hercules, CA, USA).

Protein Extraction:

Article Title: Elevated Serum Melatonin under Constant Darkness Enhances Neural Repair in Spinal Cord Injury through Regulation of Circadian Clock Proteins Expression
Article Snippet: .. Protein Extraction and Western Blotting The spinal segments were lysed in buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM NaF, 1 mM Na3 VO4 , 1% NP-40, 0.25% sodium deoxycholate, and protease inhibitor cocktail; Roche diagnostics GmbH, Mannheim, Germany). ..

Western Blot:

Article Title: Zoledronic Acid Sensitizes Renal Cell Carcinoma Cells to Radiation by Downregulating STAT1
Article Snippet: .. Western Blot Analysis Total proteins were extracted using TNESV lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 1 mM EDTA, 100 mM NaCl, 1 mM Na3 VO4 ) supplemented with complete mini protease inhibitors (Roche Applied Science, Mannheim, Germany). ..

Article Title: Elevated Serum Melatonin under Constant Darkness Enhances Neural Repair in Spinal Cord Injury through Regulation of Circadian Clock Proteins Expression
Article Snippet: .. Protein Extraction and Western Blotting The spinal segments were lysed in buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM NaF, 1 mM Na3 VO4 , 1% NP-40, 0.25% sodium deoxycholate, and protease inhibitor cocktail; Roche diagnostics GmbH, Mannheim, Germany). ..

Article Title: p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients
Article Snippet: .. Western blotting analysis Kidney tissues were lysed in RIPA buffer [50 mM Tris·HCl, pH 7.3; 150 mM NaCl; 0.1 mM EDTA; 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; 0.2% NaF; and 100 μM Na3 VO4] supplemented with complete protease inhibitors (Roche Applied Science, Indianapolis, IN). .. The kidney homogenate was centrifuged at 12,000 g for 30 min at 4°C, and the protein concentration of the supernatant was determined by the Bradford method.

Cell Culture:

Article Title: Saponin monomer 13 of dwarf lilyturf tuber (DT-13) protects serum withdrawal-induced apoptosis through PI3K/Akt in HUVEC
Article Snippet: .. After treatments, cultured HUVEC were washed with cold PBS and lysed in a buffer containing 50 mM Tris–HCl, pH 7.4, 1% NP-40, 2% SDS, 0.1% deoxycholic acid, 0.1 mM EDTA, 0.1 mM EGTA, 2.5 mM sodium pyrophosphate, 5 mM sodium fluoride, 1 mM Na3- VO4 and protease inhibitor (Roche 11836145001). .. Proteins were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA).

Lysis:

Article Title: Zoledronic Acid Sensitizes Renal Cell Carcinoma Cells to Radiation by Downregulating STAT1
Article Snippet: .. Western Blot Analysis Total proteins were extracted using TNESV lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 1 mM EDTA, 100 mM NaCl, 1 mM Na3 VO4 ) supplemented with complete mini protease inhibitors (Roche Applied Science, Mannheim, Germany). ..

Article Title: Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis
Article Snippet: .. Cell lysate was made in cell lysis buffer (20 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerol phosphate, 1 mmol/L Na3 VO4 ); and 1 mmol/L PMSF and protease inhibitor cocktail (04693116001, Roche) were added freshly. .. Protein concentration was determined by Bio-Rad Protein Assay Kit II (5000002, Hercules, CA, USA).

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  • 91
    Roche gst pak6 lysis wash buffer
    RhoD Interacts Directly with <t>Pak6</t> (A) Images showing that siRNA-mediated ablation of RhoD leads to a loss of GFP-Pak6 recruitment to the plasma membrane in cells infected with ΔF11L. In contrast, loss of Pak6 does not impact on recruitment of GFP-RhoD to the plasma membrane (see Movie S7 ). Scale bar, 5 μm. (B) Immunoblot analysis with the indicated antibodies of a GFP-Trap pull-down on cell lysates from uninfected HeLa expressing Myc-RhoD and GFP-Pak6 or GFP-RhoD and Myc-Pak6. (C) Immunoblot of glutathione-Sepharose pull-downs on HeLa cell lysates demonstrates that <t>GST-Pak6</t> interacts with RhoD, while Rhotekin preferentially associates with RhoA and RhoC. (D) Immunoblot of glutathione-Sepharose pull-downs with recombinant proteins demonstrates that GST-Pak6 interacts with RhoD but not RhoA or RhoC. See also Figure S7 .
    Gst Pak6 Lysis Wash Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche buffer a
    Cdk5 phosphorylates htt in vitro and in vivo. (A) GST and GST-tagged htt1-588 (GST-htt588) (wild-type) were purified from E. coli . Both proteins were phosphorylated by 0.1 μg of p35–cdk5 complexes. Top panel shows phosphorylated GST (lane 1) and GST-htt588 (lane 2). Bottom panel shows purified GST (lane 1) and GST-htt588 (lane 2). (B) p35–cdk5 was cotransfected to COS-7 cells. We immunoprecipitated p35–cdk5 with anti-cdk5. Httwt588 or httmu588 were pulled down with anti-Flag from distinct COS-7 cells transfected with these constructs. The figure shows in vitro kinase assays (top) and anti-Flag blot (bottom) from p35–cdk5 incubated with httwt588 and γ-[ 32 P]ATP (lane 1) and p35–cdk5 incubated with httmu588 and γ-[ 32 P]ATP (lane 2). The mixtures were resolved with 10% SDS-PAGE, and then transferred to PVDF membrane and subjected to autoradiography (top). The PVDF membrane was blotted with anti-Flag (bottom). (C) PC-12 cells were starved for 24 h, and then induced to differentiate with 100 ng/ml NGF for 48 h. Cells were treated with 20 μM of the cdk5 inhibitor roscovitine (Rosco) or DMSO (control) when cells were induced to differentiate with NGF. After 48 h of treatment, PC-12 cells were lysed in <t>buffer</t> A containing phosphatase inhibitors. Htt was immunoprecipitated with anti-htt antibody, 2166, and then detected with the antiphosphoserine antibody 16B4 (top) and anti-htt (middle). p35–cdk5 complex was pulled down from the differentiated PC-12 cells and an in vitro kinase assay was performed in the presence of DMSO (lane 1) or roscovitine (lane 2) using histone H1 as a substrate (bottom).
    Buffer A, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 1628 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche triton x 100 lysis buffer
    AMPK interacts with ULK1 directly. (A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies. (B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies. (C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies. (D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies. (E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies. (F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.
    Triton X 100 Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche immunoprecipitation buffer
    BAG3 recognizes folding status of αB-crystallin. (A) BAG3 binds to mutant type of αB-crystallin strongly. Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before <t>immunoprecipitation</t> was also loaded to confirm protein expression (right four lanes). (B) Direct recognition of mutant αB-crystallin by BAG3. Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. Mutated αB-crystallin preferentially binds to BAG3. Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).
    Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RhoD Interacts Directly with Pak6 (A) Images showing that siRNA-mediated ablation of RhoD leads to a loss of GFP-Pak6 recruitment to the plasma membrane in cells infected with ΔF11L. In contrast, loss of Pak6 does not impact on recruitment of GFP-RhoD to the plasma membrane (see Movie S7 ). Scale bar, 5 μm. (B) Immunoblot analysis with the indicated antibodies of a GFP-Trap pull-down on cell lysates from uninfected HeLa expressing Myc-RhoD and GFP-Pak6 or GFP-RhoD and Myc-Pak6. (C) Immunoblot of glutathione-Sepharose pull-downs on HeLa cell lysates demonstrates that GST-Pak6 interacts with RhoD, while Rhotekin preferentially associates with RhoA and RhoC. (D) Immunoblot of glutathione-Sepharose pull-downs with recombinant proteins demonstrates that GST-Pak6 interacts with RhoD but not RhoA or RhoC. See also Figure S7 .

    Journal: Developmental Cell

    Article Title: RhoD Inhibits RhoC-ROCK-Dependent Cell Contraction via PAK6

    doi: 10.1016/j.devcel.2017.04.010

    Figure Lengend Snippet: RhoD Interacts Directly with Pak6 (A) Images showing that siRNA-mediated ablation of RhoD leads to a loss of GFP-Pak6 recruitment to the plasma membrane in cells infected with ΔF11L. In contrast, loss of Pak6 does not impact on recruitment of GFP-RhoD to the plasma membrane (see Movie S7 ). Scale bar, 5 μm. (B) Immunoblot analysis with the indicated antibodies of a GFP-Trap pull-down on cell lysates from uninfected HeLa expressing Myc-RhoD and GFP-Pak6 or GFP-RhoD and Myc-Pak6. (C) Immunoblot of glutathione-Sepharose pull-downs on HeLa cell lysates demonstrates that GST-Pak6 interacts with RhoD, while Rhotekin preferentially associates with RhoA and RhoC. (D) Immunoblot of glutathione-Sepharose pull-downs with recombinant proteins demonstrates that GST-Pak6 interacts with RhoD but not RhoA or RhoC. See also Figure S7 .

    Article Snippet: Cells were collected by centrifugation, resuspended in GST-Pak6 lysis/wash buffer [PBS, 1% Triton-X, 50mM NaF, 1mM Na3 VO4, 1mM PMSF, protease inhibitor cocktail (Roche, UK)] and lysed by sonication.

    Techniques: Infection, Expressing, Recombinant

    Cdk5 phosphorylates htt in vitro and in vivo. (A) GST and GST-tagged htt1-588 (GST-htt588) (wild-type) were purified from E. coli . Both proteins were phosphorylated by 0.1 μg of p35–cdk5 complexes. Top panel shows phosphorylated GST (lane 1) and GST-htt588 (lane 2). Bottom panel shows purified GST (lane 1) and GST-htt588 (lane 2). (B) p35–cdk5 was cotransfected to COS-7 cells. We immunoprecipitated p35–cdk5 with anti-cdk5. Httwt588 or httmu588 were pulled down with anti-Flag from distinct COS-7 cells transfected with these constructs. The figure shows in vitro kinase assays (top) and anti-Flag blot (bottom) from p35–cdk5 incubated with httwt588 and γ-[ 32 P]ATP (lane 1) and p35–cdk5 incubated with httmu588 and γ-[ 32 P]ATP (lane 2). The mixtures were resolved with 10% SDS-PAGE, and then transferred to PVDF membrane and subjected to autoradiography (top). The PVDF membrane was blotted with anti-Flag (bottom). (C) PC-12 cells were starved for 24 h, and then induced to differentiate with 100 ng/ml NGF for 48 h. Cells were treated with 20 μM of the cdk5 inhibitor roscovitine (Rosco) or DMSO (control) when cells were induced to differentiate with NGF. After 48 h of treatment, PC-12 cells were lysed in buffer A containing phosphatase inhibitors. Htt was immunoprecipitated with anti-htt antibody, 2166, and then detected with the antiphosphoserine antibody 16B4 (top) and anti-htt (middle). p35–cdk5 complex was pulled down from the differentiated PC-12 cells and an in vitro kinase assay was performed in the presence of DMSO (lane 1) or roscovitine (lane 2) using histone H1 as a substrate (bottom).

    Journal: The Journal of Cell Biology

    Article Title: Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases

    doi: 10.1083/jcb.200412071

    Figure Lengend Snippet: Cdk5 phosphorylates htt in vitro and in vivo. (A) GST and GST-tagged htt1-588 (GST-htt588) (wild-type) were purified from E. coli . Both proteins were phosphorylated by 0.1 μg of p35–cdk5 complexes. Top panel shows phosphorylated GST (lane 1) and GST-htt588 (lane 2). Bottom panel shows purified GST (lane 1) and GST-htt588 (lane 2). (B) p35–cdk5 was cotransfected to COS-7 cells. We immunoprecipitated p35–cdk5 with anti-cdk5. Httwt588 or httmu588 were pulled down with anti-Flag from distinct COS-7 cells transfected with these constructs. The figure shows in vitro kinase assays (top) and anti-Flag blot (bottom) from p35–cdk5 incubated with httwt588 and γ-[ 32 P]ATP (lane 1) and p35–cdk5 incubated with httmu588 and γ-[ 32 P]ATP (lane 2). The mixtures were resolved with 10% SDS-PAGE, and then transferred to PVDF membrane and subjected to autoradiography (top). The PVDF membrane was blotted with anti-Flag (bottom). (C) PC-12 cells were starved for 24 h, and then induced to differentiate with 100 ng/ml NGF for 48 h. Cells were treated with 20 μM of the cdk5 inhibitor roscovitine (Rosco) or DMSO (control) when cells were induced to differentiate with NGF. After 48 h of treatment, PC-12 cells were lysed in buffer A containing phosphatase inhibitors. Htt was immunoprecipitated with anti-htt antibody, 2166, and then detected with the antiphosphoserine antibody 16B4 (top) and anti-htt (middle). p35–cdk5 complex was pulled down from the differentiated PC-12 cells and an in vitro kinase assay was performed in the presence of DMSO (lane 1) or roscovitine (lane 2) using histone H1 as a substrate (bottom).

    Article Snippet: Immunoprecipitation (IP) IP was performed using buffer A (20 mM Tris-HCl, pH 7.2, 2 mM MgCl2 , 150 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , 0.5% NP-40, and protease inhibitor cocktail [Roche]).

    Techniques: In Vitro, In Vivo, Purification, Immunoprecipitation, Transfection, Construct, Incubation, SDS Page, Autoradiography, Kinase Assay

    AMPK interacts with ULK1 directly. (A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies. (B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies. (C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies. (D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies. (E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies. (F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.

    Journal: PLoS ONE

    Article Title: The Association of AMPK with ULK1 Regulates Autophagy

    doi: 10.1371/journal.pone.0015394

    Figure Lengend Snippet: AMPK interacts with ULK1 directly. (A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies. (B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies. (C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies. (D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies. (E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies. (F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.

    Article Snippet: Immunoprecipitation and Immunoblotting Cell lysates were prepared in a 0.5% Triton X-100 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1 mM Na3 VO4 ) containing a protease inhibitor cocktail (Roche; 11873580001) and phosphatase inhibitor cocktails 1/2 (Sigma; P2850/P5726).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Infection, shRNA, Purification, Lysis

    BAG3 recognizes folding status of αB-crystallin. (A) BAG3 binds to mutant type of αB-crystallin strongly. Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before immunoprecipitation was also loaded to confirm protein expression (right four lanes). (B) Direct recognition of mutant αB-crystallin by BAG3. Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. Mutated αB-crystallin preferentially binds to BAG3. Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).

    Journal: PLoS ONE

    Article Title: BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity

    doi: 10.1371/journal.pone.0016828

    Figure Lengend Snippet: BAG3 recognizes folding status of αB-crystallin. (A) BAG3 binds to mutant type of αB-crystallin strongly. Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before immunoprecipitation was also loaded to confirm protein expression (right four lanes). (B) Direct recognition of mutant αB-crystallin by BAG3. Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. Mutated αB-crystallin preferentially binds to BAG3. Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).

    Article Snippet: 48 hours after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NAF, 2 mM Na3 VO4 , 2 mM PMSF, and 1% TritonX-100) supplemented with a mixture of protease inhibitors (CompleteTM, Roche Diagnostics).

    Techniques: Mutagenesis, SDS Page, Immunoprecipitation, Expressing, Purification, Incubation, Pull Down Assay, Staining