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Roche ip buffer
Ip Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ip buffer - by Bioz Stars, 2020-04
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Stable Transfection:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Biotin Protection Degradation Assay HEK293 cells stably expressing N-terminal FLAG-tagged FPR2/ALX, N333-stop, P342-stop, or T346-stop were grown to 100% confluency and labeled with 3 μg/ml disulfide-cleavable biotin (Pierce) for 30 min at 4 °C ( ). .. Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)).

Construct:

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: An Myc-tagged full-length Ehd3 vector was constructed using the Hpa I site of the pGA3697 vector that carried the maize Ubiquitin1 promoter and the 4x Myc coding region. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Incubation:

Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity
Article Snippet: .. For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively. .. For glutaraldehyde crosslinking, 4 μg of GFP-, Myc-RBPMS2 and Myc-RBPMS2-L410E-expressing DF1 total protein extracts were incubated at 4°C in 36 μl of 0,1% PBS-buffered glutaraldehyde solution (Sigma-Aldrich) during 10 or 30 s and reaction was stopped with 4 μl of Tris 1M pH8.

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: The cells were washed twice and incubated at 37°C for 1 h in the respective medium without FCS then followed by 3 h incubation in the same medium supplemented with 200 μCi/mL 3 H-palmitate (9,10-3 H(N)) (American Radiolabeled Chemicals, St Louis, MO). .. After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland).

Article Title: A protein secreted by the respiratory pathogen Chlamydia pneumoniae impairs IL-17 signaling via interaction with human Act1
Article Snippet: C. pneumoniae and mock infected HeLa cells at 64 hrs p.i. were washed with cold PBS and lysed in immunoprecipitation buffer ( ) containing 0.5% Triton-X-100, 20 mM Hepes (pH 7.4), 150 mM NaCl, 12.5 mM β–glycerophosphate, 1.5 mM MgCl2 , 10 mM NaF, 2 mM DTT 2 mM EGTA, 1 mM sodium orthovanadate and protease inhibitors (Roche, Indianapolis, IN). .. In experiments where chlamydial protein synthesis was inhibited, C. pneumoniae infected cells were incubated in the presence of 200 μg/ml Chloramphenicol (Sigma).

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Lysates were immunoprecipitated (anti-FLAG M2) overnight and incubated for 2 h with recombinant protein G-Sepharose (Life Technologies) and deglycosylated.

Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses
Article Snippet: Immunoblotting For Co-IP, total proteins were extracted from leaves using immunoprecipitation buffer [50mM HEPES (pH 7.5), 50mM NaCl, 10mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail (Roche, Mannheim, Germany)]; insoluble debris was pelleted by centrifuging leaf extracts at 15 000 g for 30min at 4 °C. .. The soluble protein extracts were incubated with monoclonal anti-cMyc or anti-HA agarose conjugates (Sigma-Aldrich, St Louis, MO, USA) overnight.

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
Article Snippet: After 48 h, cells (1 × 107 –2.5 × 107 ) were harvested and lysed in immunoprecipitation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% Triton-X-100, protease inhibitor cocktail tablets complete, and EDTA-free [Roche]). .. Supernatants were incubated with antibodies (1 μg) for 3 h at 4 °C, then rotated with Protein A-agarose (Millipore, CAT# 16-125) for 3 h or overnight (for endogenous protein immunoprecipitation) at 4 °C.

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: After incubation, the protoplasts were harvested and used for coimmunoprecipitation assays as previously reported, but with minor modifications ( ). .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science). .. Equivalent amounts of total proteins were incubated with anti-Flag M2 (Sigma), anti-PIAS3 monoclonal antibodies C-12 (sc-46682), or anti-EKLF monoclonal antibodies (6B3) overnight at 4 °C, followed by incubation with protein G-Sepharose for 2 h at 4 °C.

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity
Article Snippet: The lysate was incubated for 20 min on ice and centrifuged at 4°C at 10,000 × g for 10 min to remove insoluble components. .. For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche).

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: After incubation with primary Abs, the immunoblots were revealed by HRP-conjugated anti-mouse, anti-rabbit or anti-rat secondary Abs (Cell Signaling Technologies and Santa Cruz) and detection with Immobilon Western (Millipore, Billerica, MA). .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Cell Culture:

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: Whole-cell extracts of C2.7, CH3/MyoD, or C3H10T1/2 cells, cultured in GM or DM, were prepared in lysis buffer (50 mM Tris [pH 7.5], 250 mM NaCl, 1% Nonidet P-40 [NP-40], 5 mM ATP, 5 mM MgCl2 , 5 mM EDTA, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with a mixture of protease inhibitors (Roche). .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: RPE1 cells virally expressing selected plasmids were seeded in six-well plates in SILAC labelling medium supplemented with dialysed FCS and cultured over a minimum of six passages to achieve full labelling with respective isotopes and a minimum of two confluent 20 cm dishes for generation of lysates. .. Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. .. For in vitro studies, the same procedure was performed on cultured WT rat neurons aged 7, 14, 21, and 28 DIV.

Expressing:

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: For immunoprecipitation experiments, 3 × 105 C3H10T[1/2] cells were transfected with expression vectors for MyoD (pcDNA3-FLAG-MyoD) and/or CREB (RC/RSV-CREB341) at 2.5 μg each. .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: Paragraph title: Expression in mammalian cells and coimmunoprecipitation assay. ... At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice.

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Biotin Protection Degradation Assay HEK293 cells stably expressing N-terminal FLAG-tagged FPR2/ALX, N333-stop, P342-stop, or T346-stop were grown to 100% confluency and labeled with 3 μg/ml disulfide-cleavable biotin (Pierce) for 30 min at 4 °C ( ). .. Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)).

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: RPE1 cells virally expressing selected plasmids were seeded in six-well plates in SILAC labelling medium supplemented with dialysed FCS and cultured over a minimum of six passages to achieve full labelling with respective isotopes and a minimum of two confluent 20 cm dishes for generation of lysates. .. Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: .. 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science). .. Equivalent amounts of total proteins were incubated with anti-Flag M2 (Sigma), anti-PIAS3 monoclonal antibodies C-12 (sc-46682), or anti-EKLF monoclonal antibodies (6B3) overnight at 4 °C, followed by incubation with protein G-Sepharose for 2 h at 4 °C.

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: .. Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. ..

Western Blot:

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: Paragraph title: Extracts, immunoprecipitations, and Western blotting. ... Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice. .. The immunoprecipitates were analyzed by Western blotting with anti-PKR (Santa Cruz) and anti-FLAG polyclonal antibodies (Santa Cruz) ( ).

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
Article Snippet: After 48 h, cells (1 × 107 –2.5 × 107 ) were harvested and lysed in immunoprecipitation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% Triton-X-100, protease inhibitor cocktail tablets complete, and EDTA-free [Roche]). .. After being washed with lysis buffer for six times, the immunoprecipitated materials were boiled in 40 μl 2 × SDS loading buffer and subjected to western blotting (Ninety-five percent of cell extracts were used for the IP experiment, 1% of cell extracts were used as input for western blotting.).

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: Paragraph title: Cell Transfection, Protein Extraction, Immunoprecipitation, and Western Immunoblotting ... 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science).

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling
Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]). .. Immunoprecipitates were analyzed by Western blotting using rabbit anti-Flag (1:2000; Sigma) and rabbit anti-Myc (1:2000; Sigma).

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: Paragraph title: Western blot analysis and co-immunoprecipitation assay ... For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Transformation Assay:

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: Each DNA combination of all vectors was transformed into the protoplasts isolated from rice mesophyll or Oc cells. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Transfection:

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: For immunoprecipitation experiments, 3 × 105 C3H10T[1/2] cells were transfected with expression vectors for MyoD (pcDNA3-FLAG-MyoD) and/or CREB (RC/RSV-CREB341) at 2.5 μg each. .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: To detect palmitoylation, U-2 OS cells were transfected with pCMVORF3, pCMVORF3C1-4 or pCMVORF3C5-8 plasmids and Hep293TT were electroporated with full-length HEV p6 or 83–2 RNAs prior to incubation with radiolabelled palmitate 24 h or 6 days post-transfection, respectively. .. After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland).

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: .. At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice. .. The cell extract was used to immunoprecipitate FLAG-PACT with anti-FLAG (M2) agarose as described above for the in vitro interaction assay.

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
Article Snippet: Co-immunoprecipitation For protein–protein interaction analysis, A total of 4 × 106 cells were transfected with various plasmids using the PEI reagent. .. After 48 h, cells (1 × 107 –2.5 × 107 ) were harvested and lysed in immunoprecipitation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% Triton-X-100, protease inhibitor cocktail tablets complete, and EDTA-free [Roche]).

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: .. 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science). .. Equivalent amounts of total proteins were incubated with anti-Flag M2 (Sigma), anti-PIAS3 monoclonal antibodies C-12 (sc-46682), or anti-EKLF monoclonal antibodies (6B3) overnight at 4 °C, followed by incubation with protein G-Sepharose for 2 h at 4 °C.

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity
Article Snippet: HEK 293T cells were transfected using a standard calcium phosphate transfection protocol. .. For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche).

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling
Article Snippet: Different combinations of Myc-Mycbp, Myc-p66β, Flag-Sufu, Flag-Gli1, and Flag-Gli2 were transfected into HEK293T cells by Lipofectamine 2000 (Life Technologies). .. Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

Protease Inhibitor:

Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity
Article Snippet: .. For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively. .. For glutaraldehyde crosslinking, 4 μg of GFP-, Myc-RBPMS2 and Myc-RBPMS2-L410E-expressing DF1 total protein extracts were incubated at 4°C in 36 μl of 0,1% PBS-buffered glutaraldehyde solution (Sigma-Aldrich) during 10 or 30 s and reaction was stopped with 4 μl of Tris 1M pH8.

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: .. After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). .. Immunoprecipitation was carried out by the incubation of each protein lysates overnight at 4°C with 50 μL of Dynabeads Protein G (Thermo Fischer Scientific) pre-adsorbed with 1 μl of rabbit anti-ORF3 pAb, gift from Suzanne Emerson (NIH, Bethesda, MD), or unrelated rabbit serum as control, following manufacturers’s recommendations.

Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses
Article Snippet: .. Immunoblotting For Co-IP, total proteins were extracted from leaves using immunoprecipitation buffer [50mM HEPES (pH 7.5), 50mM NaCl, 10mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail (Roche, Mannheim, Germany)]; insoluble debris was pelleted by centrifuging leaf extracts at 15 000 g for 30min at 4 °C. .. The soluble protein extracts were incubated with monoclonal anti-cMyc or anti-HA agarose conjugates (Sigma-Aldrich, St Louis, MO, USA) overnight.

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: .. Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
Article Snippet: .. After 48 h, cells (1 × 107 –2.5 × 107 ) were harvested and lysed in immunoprecipitation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% Triton-X-100, protease inhibitor cocktail tablets complete, and EDTA-free [Roche]). .. Cell lysates were sonicated and centrifuged at 13,000×g for 10 min at 4 °C.

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche]. .. After brief vortexing, the samples were centrifuged at 12,000 rpm for 10 min. We added 10 μ L each of protein A and G conjugated to agarose beads (Millipore) to the supernatant for 1 h of preclearing to prevent nonspecific binding.

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: .. 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science). .. Equivalent amounts of total proteins were incubated with anti-Flag M2 (Sigma), anti-PIAS3 monoclonal antibodies C-12 (sc-46682), or anti-EKLF monoclonal antibodies (6B3) overnight at 4 °C, followed by incubation with protein G-Sepharose for 2 h at 4 °C.

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity
Article Snippet: .. For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche). .. The immunoprecipitation of the supernatants, both 293T and total brain, was performed with Dynabeads (Invitrogen) following the manufacturer's instructions.

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: .. Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. ..

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling
Article Snippet: .. Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]). .. The supernatant was removed and bound to 20 μL of anti-Flag M2 beads (Sigma) or anti-Myc beads (Santa Cruz Biotechnology) overnight at 4°C with constant nutation.

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche). .. The extracts (600 μg proteins) were incubated with rabbit monoclonal anti-Src Ab(Abcam) or rabbit IgG for 12 h at 4°C.

Infection:

Article Title: A protein secreted by the respiratory pathogen Chlamydia pneumoniae impairs IL-17 signaling via interaction with human Act1
Article Snippet: .. C. pneumoniae and mock infected HeLa cells at 64 hrs p.i. were washed with cold PBS and lysed in immunoprecipitation buffer ( ) containing 0.5% Triton-X-100, 20 mM Hepes (pH 7.4), 150 mM NaCl, 12.5 mM β–glycerophosphate, 1.5 mM MgCl2 , 10 mM NaF, 2 mM DTT 2 mM EGTA, 1 mM sodium orthovanadate and protease inhibitors (Roche, Indianapolis, IN). .. In experiments where chlamydial protein synthesis was inhibited, C. pneumoniae infected cells were incubated in the presence of 200 μg/ml Chloramphenicol (Sigma).

Sonication:

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
Article Snippet: After 48 h, cells (1 × 107 –2.5 × 107 ) were harvested and lysed in immunoprecipitation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% Triton-X-100, protease inhibitor cocktail tablets complete, and EDTA-free [Roche]). .. Cell lysates were sonicated and centrifuged at 13,000×g for 10 min at 4 °C.

Recombinant:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Lysates were immunoprecipitated (anti-FLAG M2) overnight and incubated for 2 h with recombinant protein G-Sepharose (Life Technologies) and deglycosylated.

Radioactivity:

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: Paragraph title: Radiolabeling and immunoprecipitation ... After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland).

Mutagenesis:

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: HT1080 cells were transfected in 100-mm culture dishes with 10 μg of total DNA (5 μg of CMV-PKR [K296R] and 5 μg of FLAG-PACT mutant DNA) using the Lipofectamine reagent (Gibco-BRL). .. At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice.

Isolation:

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: Each DNA combination of all vectors was transformed into the protoplasts isolated from rice mesophyll or Oc cells. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: Western blot analysis and co-immunoprecipitation assay Whole cell, cytosolic, or nuclear lysates were isolated as previously described [ ]. .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Labeling:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Biotin Protection Degradation Assay HEK293 cells stably expressing N-terminal FLAG-tagged FPR2/ALX, N333-stop, P342-stop, or T346-stop were grown to 100% confluency and labeled with 3 μg/ml disulfide-cleavable biotin (Pierce) for 30 min at 4 °C ( ). .. Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)).

Mouse Assay:

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: .. Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. ..

Protein Extraction:

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: Paragraph title: Cell Transfection, Protein Extraction, Immunoprecipitation, and Western Immunoblotting ... 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science).

Degradation Assay:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Paragraph title: Biotin Protection Degradation Assay ... Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)).

Lysis:

Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity
Article Snippet: Immunoprecipitation and glutaraldehyde crosslinking DF-1 cells were lysed in lysis buffer (20-mM Tris pH8, 50-mM NaCl, 1% NP40, cOmplete ethylenediaminetetraacetic acid (EDTA)-free Protease Inhibitor Cocktail (Roche)). .. For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively.

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: Whole-cell extracts of C2.7, CH3/MyoD, or C3H10T1/2 cells, cultured in GM or DM, were prepared in lysis buffer (50 mM Tris [pH 7.5], 250 mM NaCl, 1% Nonidet P-40 [NP-40], 5 mM ATP, 5 mM MgCl2 , 5 mM EDTA, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with a mixture of protease inhibitors (Roche). .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors.

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
Article Snippet: After 48 h, cells (1 × 107 –2.5 × 107 ) were harvested and lysed in immunoprecipitation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% Triton-X-100, protease inhibitor cocktail tablets complete, and EDTA-free [Roche]). .. After being washed with lysis buffer for six times, the immunoprecipitated materials were boiled in 40 μl 2 × SDS loading buffer and subjected to western blotting (Ninety-five percent of cell extracts were used for the IP experiment, 1% of cell extracts were used as input for western blotting.).

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science). .. Immunoprecipitates were washed four times with lysis buffer and then subjected to Western blot analyses utilizing: anti-Flag M2-HRP antibody (catalog no. A8592; Sigma); anti-EKLF 7B2, 4B9, and 6B3 antibodies (mouse monoclonal antibodies made in this laboratory) ( , ); anti-EKLF goat polyclonal antibody F-20 (catalog no. sc-27194; Santa Cruz Biotechnology); anti-PIAS3 E-3 antibody (catalog no. sc-48339; Santa Cruz Biotechnology); and anti-Hsp90 H-114 antibody (sc-7947) (Santa Cruz Biotechnology).

Chloramphenicol Acetyltransferase Assay:

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
Article Snippet: After 48 h, cells (1 × 107 –2.5 × 107 ) were harvested and lysed in immunoprecipitation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% Triton-X-100, protease inhibitor cocktail tablets complete, and EDTA-free [Roche]). .. Supernatants were incubated with antibodies (1 μg) for 3 h at 4 °C, then rotated with Protein A-agarose (Millipore, CAT 16-125) for 3 h or overnight (for endogenous protein immunoprecipitation) at 4 °C.

SDS Page:

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). .. Samples were then incubated for 5 min at 90°C in Laemmli buffer and separated onto a 17% SDS-PAGE.

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Samples were resolved by SDS-PAGE and visualized with streptavidin overlay (VECTASTAIN ABC immunoperoxidase reagent, Vector Laboratories).

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: The lysates (60 μg each) were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Plasmid Preparation:

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Samples were resolved by SDS-PAGE and visualized with streptavidin overlay (VECTASTAIN ABC immunoperoxidase reagent, Vector Laboratories).

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: An Myc-tagged full-length Ehd3 vector was constructed using the Hpa I site of the pGA3697 vector that carried the maize Ubiquitin1 promoter and the 4x Myc coding region. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: .. 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science). .. Equivalent amounts of total proteins were incubated with anti-Flag M2 (Sigma), anti-PIAS3 monoclonal antibodies C-12 (sc-46682), or anti-EKLF monoclonal antibodies (6B3) overnight at 4 °C, followed by incubation with protein G-Sepharose for 2 h at 4 °C.

Co-Immunoprecipitation Assay:

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: Paragraph title: Expression in mammalian cells and coimmunoprecipitation assay. ... At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice.

Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses
Article Snippet: .. Immunoblotting For Co-IP, total proteins were extracted from leaves using immunoprecipitation buffer [50mM HEPES (pH 7.5), 50mM NaCl, 10mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail (Roche, Mannheim, Germany)]; insoluble debris was pelleted by centrifuging leaf extracts at 15 000 g for 30min at 4 °C. .. The soluble protein extracts were incubated with monoclonal anti-cMyc or anti-HA agarose conjugates (Sigma-Aldrich, St Louis, MO, USA) overnight.

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: Paragraph title: Western blot analysis and co-immunoprecipitation assay ... For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche).

Binding Assay:

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche]. .. After brief vortexing, the samples were centrifuged at 12,000 rpm for 10 min. We added 10 μ L each of protein A and G conjugated to agarose beads (Millipore) to the supernatant for 1 h of preclearing to prevent nonspecific binding.

In Vitro:

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice. .. The cell extract was used to immunoprecipitate FLAG-PACT with anti-FLAG (M2) agarose as described above for the in vitro interaction assay.

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. .. For in vitro studies, the same procedure was performed on cultured WT rat neurons aged 7, 14, 21, and 28 DIV.

Spectrophotometry:

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

Produced:

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: HA-tagged vectors were produced with the Hpa I and Kpn I sites of the pGA3698 vector, which contained the maize Ubiquitin1 promoter and the 3x HA coding region. .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche].

Immunoprecipitation:

Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity
Article Snippet: .. For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively. .. For glutaraldehyde crosslinking, 4 μg of GFP-, Myc-RBPMS2 and Myc-RBPMS2-L410E-expressing DF1 total protein extracts were incubated at 4°C in 36 μl of 0,1% PBS-buffered glutaraldehyde solution (Sigma-Aldrich) during 10 or 30 s and reaction was stopped with 4 μl of Tris 1M pH8.

Article Title: MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway †
Article Snippet: .. Cell extracts were prepared in 0.5 ml of immunoprecipitation buffer (50 mM Tris [pH 8], 120 mM NaCl, 0.5% NP-40, 5 mM β-glycerophosphate, 0.1 mM Na3 VO4 , 10 mM NaF) supplemented with Roche protease inhibitors. ..

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Article Snippet: .. After three washes with cold PBS, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl pH 7.4; 1 mM EDTA; 150 mM NaCl; 1% Triton X-100) supplemented with 1X cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). .. Immunoprecipitation was carried out by the incubation of each protein lysates overnight at 4°C with 50 μL of Dynabeads Protein G (Thermo Fischer Scientific) pre-adsorbed with 1 μl of rabbit anti-ORF3 pAb, gift from Suzanne Emerson (NIH, Bethesda, MD), or unrelated rabbit serum as control, following manufacturers’s recommendations.

Article Title: A protein secreted by the respiratory pathogen Chlamydia pneumoniae impairs IL-17 signaling via interaction with human Act1
Article Snippet: .. C. pneumoniae and mock infected HeLa cells at 64 hrs p.i. were washed with cold PBS and lysed in immunoprecipitation buffer ( ) containing 0.5% Triton-X-100, 20 mM Hepes (pH 7.4), 150 mM NaCl, 12.5 mM β–glycerophosphate, 1.5 mM MgCl2 , 10 mM NaF, 2 mM DTT 2 mM EGTA, 1 mM sodium orthovanadate and protease inhibitors (Roche, Indianapolis, IN). .. In experiments where chlamydial protein synthesis was inhibited, C. pneumoniae infected cells were incubated in the presence of 200 μg/ml Chloramphenicol (Sigma).

Article Title: Modular Structure of PACT: Distinct Domains for Binding and Activating PKR
Article Snippet: .. At 24 h after transfection, cells were lysed in immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1 mM DTT, 100 mM NaCl, 2 mM MgCl2 , complete protease inhibitors [Roche], 20% glycerol) on ice. .. The cell extract was used to immunoprecipitate FLAG-PACT with anti-FLAG (M2) agarose as described above for the in vitro interaction assay.

Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Article Snippet: .. Cells were then placed in 5 ml of medium stimulated for 30, 90, or 180 min. All plates (except the 100%) were then washed in PBS, stripped (50 mm glutathione, 0.3 mm NaCl, 75 mm NaOH, 1% FBS) at 4 °C for 30 min (to remove remaining cell surface-biotinylated receptors), quenched (PBS containing 1 mm iodoacetamide, 0.1% BSA), and then lysed in immunoprecipitation buffer (containing 0.1% Triton X-100, 150 mm NaCl, 25 mm KCl, 10 mm Tris-HCl (pH 7.4), and protease inhibitors (Roche Applied Science)). .. Lysates were immunoprecipitated (anti-FLAG M2) overnight and incubated for 2 h with recombinant protein G-Sepharose (Life Technologies) and deglycosylated.

Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses
Article Snippet: .. Immunoblotting For Co-IP, total proteins were extracted from leaves using immunoprecipitation buffer [50mM HEPES (pH 7.5), 50mM NaCl, 10mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail (Roche, Mannheim, Germany)]; insoluble debris was pelleted by centrifuging leaf extracts at 15 000 g for 30min at 4 °C. .. The soluble protein extracts were incubated with monoclonal anti-cMyc or anti-HA agarose conjugates (Sigma-Aldrich, St Louis, MO, USA) overnight.

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: .. Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
Article Snippet: .. After 48 h, cells (1 × 107 –2.5 × 107 ) were harvested and lysed in immunoprecipitation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% Triton-X-100, protease inhibitor cocktail tablets complete, and EDTA-free [Roche]). .. Cell lysates were sonicated and centrifuged at 13,000×g for 10 min at 4 °C.

Article Title: Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date3 1 [W] [OPEN]
Article Snippet: .. Briefly, the protoplasts were resuspended in Immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 1 m m EDTA, 150 m m sodium chloride, 1% [v/v] Triton X-100, 1 m m dithiothreitol, 2 m m NaF, 50 μ m MG132, and an adequate amount of Protease inhibitor cocktail [Roche]. .. After brief vortexing, the samples were centrifuged at 12,000 rpm for 10 min. We added 10 μ L each of protein A and G conjugated to agarose beads (Millipore) to the supernatant for 1 h of preclearing to prevent nonspecific binding.

Article Title: Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3) *
Article Snippet: .. 293T cells were transiently transfected with the expression plasmid as indicated using FuGENE 6 (Promega) reagent for 36 h. Cells were lysed with immunoprecipitation buffer containing: 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1% Nonidet P-40, 5 m m EDTA, 10 μg/ml leupeptin, 5 m m sodium fluoride, 1 m m sodium vanadate, supplemented with complete protease inhibitor mixture (Roche Applied Science). .. Equivalent amounts of total proteins were incubated with anti-Flag M2 (Sigma), anti-PIAS3 monoclonal antibodies C-12 (sc-46682), or anti-EKLF monoclonal antibodies (6B3) overnight at 4 °C, followed by incubation with protein G-Sepharose for 2 h at 4 °C.

Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity
Article Snippet: .. For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche). .. The immunoprecipitation of the supernatants, both 293T and total brain, was performed with Dynabeads (Invitrogen) following the manufacturer's instructions.

Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK
Article Snippet: .. Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C. ..

Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling
Article Snippet: .. Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]). .. The supernatant was removed and bound to 20 μL of anti-Flag M2 beads (Sigma) or anti-Myc beads (Santa Cruz Biotechnology) overnight at 4°C with constant nutation.

Article Title: Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation
Article Snippet: .. For immunoprecipitation, cell extracts were prepared in immunoprecipitation buffer (10 mM HEPES [pH 7.6], 15 mM KCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM PMSF) and complete protease inhibitor (Roche). .. The extracts (600 μg proteins) were incubated with rabbit monoclonal anti-Src Ab(Abcam) or rabbit IgG for 12 h at 4°C.

Staining:

Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). .. Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer ( ; ; , ; ).

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  • 96
    Roche immunoprecipitation buffer
    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of <t>co-immunoprecipitation</t> experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
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    Image Search Results


    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Journal: Molecular psychiatry

    Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

    doi: 10.1038/s41380-018-0027-3

    Figure Lengend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C.

    Techniques: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

    Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.

    Journal: Journal of Cell Science

    Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin

    doi: 10.1242/jcs.211672

    Figure Lengend Snippet: Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.

    Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

    Techniques: Immunoprecipitation, Generated, Transfection, Isolation, Western Blot, Binding Assay, Mutagenesis, Construct

    Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. (A) HEK293-T cells were transiently transfected with constructs encoding GFP, GFP-SNX20, GFP-SNX21 and various SNX21 point mutants. After GFP-nanotrap immunoisolation, precipitates were analysed by SDS-PAGE and western blotting. GFP-SNX21 precipitates both septin 2 and septin 7, an interaction that occurs via the SNX21 PXB domain and appears to require the endosomal localisation of SNX21. Data are representative of three biological replicates. (B) Amino acid residues mutated in the current study mapped onto the published structure of the mouse SNX21 ( Clairfeuille et al., 2015 ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.

    Journal: Journal of Cell Science

    Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin

    doi: 10.1242/jcs.211672

    Figure Lengend Snippet: Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. (A) HEK293-T cells were transiently transfected with constructs encoding GFP, GFP-SNX20, GFP-SNX21 and various SNX21 point mutants. After GFP-nanotrap immunoisolation, precipitates were analysed by SDS-PAGE and western blotting. GFP-SNX21 precipitates both septin 2 and septin 7, an interaction that occurs via the SNX21 PXB domain and appears to require the endosomal localisation of SNX21. Data are representative of three biological replicates. (B) Amino acid residues mutated in the current study mapped onto the published structure of the mouse SNX21 ( Clairfeuille et al., 2015 ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.

    Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

    Techniques: Immunoprecipitation, Transfection, Construct, SDS Page, Western Blot, Mutagenesis

    AKIN10 interacts directly with EIN3. ( a ) Structure modeling of protein kinase domain of Snf1 in S . pombe and AKIN10 in A . thaliana . Individual protein structures were generated using SWISS-MODEL ( http://swissmodel.expasy.org ) and visualized in a superimposed image using PyMOL ( http://www.pymol.org ). ( b ) Binary protein-protein interaction of AKIN10 and EIN3 was analyzed using a yeast two-hybrid system. ( c ) Protein-protein interaction of AKIN10 and EIN3 was confirmed by co-immunoprecipitation using Arabidopsis protoplasts transfected with a combination of AKIN10 and EIN3 constructs. ( d ) Binary protein-protein interactions between AKIN10 with MYB2, MYC3, or MYC4 were analyzed using a yeast two-hybrid system. ( e ) AKIN10-dependent EIN3 phosphorylation in vitro was shown with GST-EIN3 fragments as substrate. Coomassie blue staining was used for protein substrate visualization. All experiments were repeated with consistent results.

    Journal: Scientific Reports

    Article Title: Regulatory Functions of Cellular Energy Sensor SNF1-Related Kinase1 for Leaf Senescence Delay through ETHYLENE- INSENSITIVE3 Repression

    doi: 10.1038/s41598-017-03506-1

    Figure Lengend Snippet: AKIN10 interacts directly with EIN3. ( a ) Structure modeling of protein kinase domain of Snf1 in S . pombe and AKIN10 in A . thaliana . Individual protein structures were generated using SWISS-MODEL ( http://swissmodel.expasy.org ) and visualized in a superimposed image using PyMOL ( http://www.pymol.org ). ( b ) Binary protein-protein interaction of AKIN10 and EIN3 was analyzed using a yeast two-hybrid system. ( c ) Protein-protein interaction of AKIN10 and EIN3 was confirmed by co-immunoprecipitation using Arabidopsis protoplasts transfected with a combination of AKIN10 and EIN3 constructs. ( d ) Binary protein-protein interactions between AKIN10 with MYB2, MYC3, or MYC4 were analyzed using a yeast two-hybrid system. ( e ) AKIN10-dependent EIN3 phosphorylation in vitro was shown with GST-EIN3 fragments as substrate. Coomassie blue staining was used for protein substrate visualization. All experiments were repeated with consistent results.

    Article Snippet: co-Immunoprecipitation and Protein immunoblotting Total protein was extracted from the transfected protoplasts using immunoprecipitation (IP) buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% Triton-X100, and Complete™ protease inhibitors (Roche, Basel, Switzerland).

    Techniques: Generated, Immunoprecipitation, Transfection, Construct, In Vitro, Staining

    Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Expressing

    A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Staining, Immunoprecipitation, Mass Spectrometry, SDS Page, Binding Assay, Western Blot, Expressing, Derivative Assay, Immunofluorescence, Transfection, Transduction, Construct