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    Nonidet P 40 Substitute solution
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    Millipore np40 detergent
    Nonidet P 40 Substitute solution

    https://www.bioz.com/result/np40 detergent/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    np40 detergent - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum"

    Article Title: Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002052

    hd − cells produced spores with reduced viability. The viability of wild-type ( grey bars ) and hd − ( black bars ) spores was assessed. Spores were untreated, heated to 45°C for 10 minutes or incubated with 0.5% NP40 detergent for 5 minutes and aliquots of 100 spores were plated in triplicate onto SM-5 agar plates in a suspension of bacteria and grown for 7 days at 21°C. The relative viability of hd − spores was assessed by counting the number of clear plaques formed on the bacterial lawns. Results are representative of three independent experiments.
    Figure Legend Snippet: hd − cells produced spores with reduced viability. The viability of wild-type ( grey bars ) and hd − ( black bars ) spores was assessed. Spores were untreated, heated to 45°C for 10 minutes or incubated with 0.5% NP40 detergent for 5 minutes and aliquots of 100 spores were plated in triplicate onto SM-5 agar plates in a suspension of bacteria and grown for 7 days at 21°C. The relative viability of hd − spores was assessed by counting the number of clear plaques formed on the bacterial lawns. Results are representative of three independent experiments.

    Techniques Used: Produced, Incubation

    2) Product Images from "Crude subcellular fractionation of cultured mammalian cell lines"

    Article Title: Crude subcellular fractionation of cultured mammalian cell lines

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-2-243

    Optimization of digitonin concentration for the extraction of cytosolic proteins . (A) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well plate and were harvested 48 hours later. Cells were then lysed in 400 μl of buffer containing digitonin at the concentration indicated. Following centrifugation and collection of the supernatant the remaining cell pellet was further extracted in NP40 lysis buffer. An aliquot of each extract was then analysed by 4-12% SDS PAGE (Invitrogen #NP0322) followed by staining with Coomassie blue. (B) The concentration of protein in each extract was determined by colorimetric protein assay (Pierce #23232) and the results were plotted to demonstrate that total protein extracted was the same regardless of the starting concentration of digitonin. (C) Extracted proteins from each fraction were analyzed by Western blot using an anti GAPDH antibody (NOVUS Biologicals #300-221B) as a marker of the cytosol and an anti BiP antibody (SIGMA # G918) as a marker of the endoplasmic reticulum.
    Figure Legend Snippet: Optimization of digitonin concentration for the extraction of cytosolic proteins . (A) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well plate and were harvested 48 hours later. Cells were then lysed in 400 μl of buffer containing digitonin at the concentration indicated. Following centrifugation and collection of the supernatant the remaining cell pellet was further extracted in NP40 lysis buffer. An aliquot of each extract was then analysed by 4-12% SDS PAGE (Invitrogen #NP0322) followed by staining with Coomassie blue. (B) The concentration of protein in each extract was determined by colorimetric protein assay (Pierce #23232) and the results were plotted to demonstrate that total protein extracted was the same regardless of the starting concentration of digitonin. (C) Extracted proteins from each fraction were analyzed by Western blot using an anti GAPDH antibody (NOVUS Biologicals #300-221B) as a marker of the cytosol and an anti BiP antibody (SIGMA # G918) as a marker of the endoplasmic reticulum.

    Techniques Used: Concentration Assay, Centrifugation, Lysis, SDS Page, Staining, Western Blot, Marker

    Demonstration of the advantages of crude fractionation over whole cell lysis and scale up of the approach . (A) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well plate and subsequently lysed directly in NP40 or RIPA lysis buffer or processed according to the protocol in Figure 2 (Sequential). An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue or by Western blotting and probing with antibodies to various marker proteins of specific intracellular organelles as previously. (B) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well dish and then transfected the next day in triplicate with 400 ng of a construct encoding V5/6×-HIS tagged ERGIC-53. Cells were extracted 48 hours later either directly in RIPA buffer, directly in NP40 buffer or according to our extraction protocol. Proteins were then partially purified from each extract using Nickel resin (SIGMA #P6611) and eluted proteins were analyzed by 10% SDS PAGE followed by staining with Coomassie blue or by Western blotting and probing with the anti V5 antibody. (C) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well dish and then transfected the next day in with 1600 ng of pcDNA6/V5-His (Invitrogen) or constructs encoding WT or MUT COMP. Cells were harvested 48 hours later and extracted according to our protocol. (D) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well dish or 8.4 × 10 5 cells per well of a 35 mm dish and processed according to the protocol in Figure 2. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting and probing with antibodies to GAPDH, BiP and Lamin A.
    Figure Legend Snippet: Demonstration of the advantages of crude fractionation over whole cell lysis and scale up of the approach . (A) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well plate and subsequently lysed directly in NP40 or RIPA lysis buffer or processed according to the protocol in Figure 2 (Sequential). An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue or by Western blotting and probing with antibodies to various marker proteins of specific intracellular organelles as previously. (B) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well dish and then transfected the next day in triplicate with 400 ng of a construct encoding V5/6×-HIS tagged ERGIC-53. Cells were extracted 48 hours later either directly in RIPA buffer, directly in NP40 buffer or according to our extraction protocol. Proteins were then partially purified from each extract using Nickel resin (SIGMA #P6611) and eluted proteins were analyzed by 10% SDS PAGE followed by staining with Coomassie blue or by Western blotting and probing with the anti V5 antibody. (C) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well dish and then transfected the next day in with 1600 ng of pcDNA6/V5-His (Invitrogen) or constructs encoding WT or MUT COMP. Cells were harvested 48 hours later and extracted according to our protocol. (D) HEK293 cells were plated at a density of 4 × 10 5 cells per well of a 12 well dish or 8.4 × 10 5 cells per well of a 35 mm dish and processed according to the protocol in Figure 2. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting and probing with antibodies to GAPDH, BiP and Lamin A.

    Techniques Used: Fractionation, Lysis, SDS Page, Staining, Western Blot, Marker, Transfection, Construct, Purification

    3) Product Images from "Evidence for allosteric effects on p53 oligomerization induced by phosphorylation"

    Article Title: Evidence for allosteric effects on p53 oligomerization induced by phosphorylation

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.3344

    Phosphorylated p53 shows increased reactivity with conformation‐specific PAb1620 monoclonal antibodies. Samples were phosphorylated in vitro and serially diluted in NP40 buffer. Samples were captured with PAb1620 monoclonal antibodies and subsequently detected with DO‐1‐HRP conjugate antibody. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP.
    Figure Legend Snippet: Phosphorylated p53 shows increased reactivity with conformation‐specific PAb1620 monoclonal antibodies. Samples were phosphorylated in vitro and serially diluted in NP40 buffer. Samples were captured with PAb1620 monoclonal antibodies and subsequently detected with DO‐1‐HRP conjugate antibody. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP.

    Techniques Used: In Vitro, Protein Concentration, Activity Assay

    Phosphorylated p53 forms oligomers and can be detected by two‐site ELISA. (A) Schematic of two‐site ELISA detection of p53 tetramers. In the monomeric state, the N‐terminal region of p53 is captured and blocked by DO‐1. The addition of the kinase and phosphate donor pair causes p53 to form tetramers. These oligomers have exposed N‐terminal regions which can be detected by DO‐1‐HRP conjugated antibodies. (B) Samples were phosphorylated in vitro and serially diluted 10 times in NP40 buffer. Samples were captured by DO‐1 monoclonal antibody and subsequently detected with DO‐1‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP. (C) The samples were analyzed using blue native electrophoresis. The proteins transferred on membranes by western blotting were detected by DO1 antibody, phosphorylated Ser392 was detected by FP3 monoclonal antibody. Arrows labeled M, D, T indicate monomers dimers and tetramers, respectively.
    Figure Legend Snippet: Phosphorylated p53 forms oligomers and can be detected by two‐site ELISA. (A) Schematic of two‐site ELISA detection of p53 tetramers. In the monomeric state, the N‐terminal region of p53 is captured and blocked by DO‐1. The addition of the kinase and phosphate donor pair causes p53 to form tetramers. These oligomers have exposed N‐terminal regions which can be detected by DO‐1‐HRP conjugated antibodies. (B) Samples were phosphorylated in vitro and serially diluted 10 times in NP40 buffer. Samples were captured by DO‐1 monoclonal antibody and subsequently detected with DO‐1‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP. (C) The samples were analyzed using blue native electrophoresis. The proteins transferred on membranes by western blotting were detected by DO1 antibody, phosphorylated Ser392 was detected by FP3 monoclonal antibody. Arrows labeled M, D, T indicate monomers dimers and tetramers, respectively.

    Techniques Used: Enzyme-linked Immunosorbent Assay, In Vitro, Protein Concentration, Activity Assay, Electrophoresis, Western Blot, Labeling

    Samples of purified p53 protein were phosphorylated in vitro by CK2 and ATP. (A) Samples were phosphorylated in vitro , subjected to electrophoresis on a 4–16% (w/v) SDS‐polyacrylamide gel, and visualized by immuno‐blotting with DO‐1 and FP3 monoclonal antibodies at 1 μg/mL. (B) Samples were phosphorylated in vitro and titrated 10 times in a NP40 buffer for two‐site ELISA detection. Samples were captured by DO‐1 monoclonal antibody and subsequently detected with FP3‐HRP conjugated antibody. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP.
    Figure Legend Snippet: Samples of purified p53 protein were phosphorylated in vitro by CK2 and ATP. (A) Samples were phosphorylated in vitro , subjected to electrophoresis on a 4–16% (w/v) SDS‐polyacrylamide gel, and visualized by immuno‐blotting with DO‐1 and FP3 monoclonal antibodies at 1 μg/mL. (B) Samples were phosphorylated in vitro and titrated 10 times in a NP40 buffer for two‐site ELISA detection. Samples were captured by DO‐1 monoclonal antibody and subsequently detected with FP3‐HRP conjugated antibody. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as percentage of the maximal value obtained. (○) p53 only; (◼) p53 + CK2; (▲) p53 + ATP; (×) p53 + CK2/ATP.

    Techniques Used: Purification, In Vitro, Electrophoresis, Enzyme-linked Immunosorbent Assay, Protein Concentration, Activity Assay

    CK2 binding to p53 blocks the Bp53‐10 epitope within the C‐terminal region of p53. p53 was incubated with either 500 units (A) or 5 units (B) of CK2 and ATP. Samples were phosphorylated in vitro and serially diluted ten times in NP40 buffer. Samples were captured with DO‐1 monoclonal antibody and subsequently detected with Bp53‐10‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as a percentage of the maximal value obtained.
    Figure Legend Snippet: CK2 binding to p53 blocks the Bp53‐10 epitope within the C‐terminal region of p53. p53 was incubated with either 500 units (A) or 5 units (B) of CK2 and ATP. Samples were phosphorylated in vitro and serially diluted ten times in NP40 buffer. Samples were captured with DO‐1 monoclonal antibody and subsequently detected with Bp53‐10‐HRP conjugate. Protein concentration on the x‐axis is plotted on a semilogarithmic scale against HRP activity (absorbance at 450 nm) on the y‐axis, plotted as a percentage of the maximal value obtained.

    Techniques Used: Binding Assay, Incubation, In Vitro, Protein Concentration, Activity Assay

    Related Articles

    Lysis:

    Article Title: Association of Phosphorylated Serine/Arginine (SR) Splicing Factors With The U1-Small Ribonucleoprotein (snRNP) Autoantigen Complex Accompanies Apoptotic Cell Death
    Article Snippet: .. After the third NP-40 lysis buffer wash, the immunoprecipitate was digested in a volume of 300 μl for 1 h at 37°C in a solution containing 50 μg/ml proteinase K ( Sigma Chemical Co. ), 10 mM Tris, pH 7.8, 10 mM EDTA, and 0.5% SDS. .. The RNA was isolated after two extractions with a phenol/chloroform/isoamyl alcohol (25:24:1) mixture ( GIBCO BRL ).

    Article Title: Crude subcellular fractionation of cultured mammalian cell lines
    Article Snippet: .. Briefly, following the NP40 lysis step the nucleus was solubilized and its contents released using RIPA buffer supplemented with Benzonase (SIGMA) to digest DNA and RNA. ..

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: Immunoblotting analysis and coimmunoprecipitation (coIP) For immunoblotting, cells were treated by H2 O2 exposure or amino acid starvation (amino acid-free Earle's Balanced Salt Solution, HyClone, SH30029.02) for different time periods, and then lysed for immunoblotting analysis according to standard protocol. .. For CoIP assay, plasmids were transfected into HeLa cells prior to different treatment and the cells were lysed by NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) and subjected for immunoprecipitation analysis according to the manufacturer's protocol of Sigma anti-Flag M2 affinity gel (Sigma, A2220). .. Electron microscopy The cells were fixed using 2.5% glutaraldehyde and 100 mM phosphate buffer, pH 7.2, one to 1.5 h at room temperature.

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: HEK293 cells in a 10-cm dish were transfected with a pShuttle-CMV expression vector for Flag-tagged hHSF1 or an hHSF1 point mutant, and were lysed with NP-40 lysis buffer. .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Article Title: Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It
    Article Snippet: RAW264.7 cells were stimulated with 200 U/ml IFN-γ overnight, starved for 1 h, metabolically labeled with [35 S] for 10 min, and chased for the indicated time. .. At the end of each time point, cells were lysed in 0.5% NP-40 lysis buffer and IPs and re-IPs were performed with a rabbit polyclonal anti-mGBP1 antiserum and a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich). ..

    Incubation:

    Article Title: Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum
    Article Snippet: Spores from mature fruiting bodies were harvested using sterile pipette tips containing 10 µL of spore buffer (40 mM KH2 PO4 , 20 mM KCl, 2.5 mM MgCl2 ), washed twice by centrifugation at 12,500 g for 2 minutes at room temperature and counted with a hemocytometer. .. Aliquots of 100 spores were heated to 45°C for 10 minutes, incubated with 0.5% NP40 detergent (Sigma-Aldrich, St Louis, MO) for 5 minutes or incubated with spore buffer alone for 5 minutes as a control. ..

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: HEK293 cells in a 10-cm dish were transfected with a pShuttle-CMV expression vector for Flag-tagged hHSF1 or an hHSF1 point mutant, and were lysed with NP-40 lysis buffer. .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Co-Immunoprecipitation Assay:

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: Immunoblotting analysis and coimmunoprecipitation (coIP) For immunoblotting, cells were treated by H2 O2 exposure or amino acid starvation (amino acid-free Earle's Balanced Salt Solution, HyClone, SH30029.02) for different time periods, and then lysed for immunoblotting analysis according to standard protocol. .. For CoIP assay, plasmids were transfected into HeLa cells prior to different treatment and the cells were lysed by NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) and subjected for immunoprecipitation analysis according to the manufacturer's protocol of Sigma anti-Flag M2 affinity gel (Sigma, A2220). .. Electron microscopy The cells were fixed using 2.5% glutaraldehyde and 100 mM phosphate buffer, pH 7.2, one to 1.5 h at room temperature.

    Transfection:

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: Immunoblotting analysis and coimmunoprecipitation (coIP) For immunoblotting, cells were treated by H2 O2 exposure or amino acid starvation (amino acid-free Earle's Balanced Salt Solution, HyClone, SH30029.02) for different time periods, and then lysed for immunoblotting analysis according to standard protocol. .. For CoIP assay, plasmids were transfected into HeLa cells prior to different treatment and the cells were lysed by NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) and subjected for immunoprecipitation analysis according to the manufacturer's protocol of Sigma anti-Flag M2 affinity gel (Sigma, A2220). .. Electron microscopy The cells were fixed using 2.5% glutaraldehyde and 100 mM phosphate buffer, pH 7.2, one to 1.5 h at room temperature.

    Article Title: Epstein-Barr Virus Nuclear Protein 2 Has at Least Two N-Terminal Domains That Mediate Self-Association
    Article Snippet: The interaction of the Flag epitope-tagged EBNA-2 amino-terminal 194-amino-acid fusion protein (F194) with wild-type EBNA-2 was then investigated by transfection of BJAB cells with pSGF194 and with pSG5 expressing wild-type EBNA-2 (pSGWE2). .. After 24 h, the transfected BJAB cells were lysed in 1% NP-40 buffer (10 mM Tris-Cl [pH 7.4], 1 mM EDTA, 150 mM NaCl, 3% glycerol, 1 mM phenylmethylsulfonyl fluoride, 5-μg/ml leupeptin, 10-μg/ml aprotinin), and half was used to immunoprecipitate F194 with M2 anti-Flag monoclonal antibody (Sigma Chemical Co.) and protein G-Sepharose (Pharmacia Corporation), while the other half was used to immunoprecipitate wild-type EBNA-2 with PE2 monoclonal antibody. ..

    Immunoprecipitation:

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: Immunoblotting analysis and coimmunoprecipitation (coIP) For immunoblotting, cells were treated by H2 O2 exposure or amino acid starvation (amino acid-free Earle's Balanced Salt Solution, HyClone, SH30029.02) for different time periods, and then lysed for immunoblotting analysis according to standard protocol. .. For CoIP assay, plasmids were transfected into HeLa cells prior to different treatment and the cells were lysed by NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) and subjected for immunoprecipitation analysis according to the manufacturer's protocol of Sigma anti-Flag M2 affinity gel (Sigma, A2220). .. Electron microscopy The cells were fixed using 2.5% glutaraldehyde and 100 mM phosphate buffer, pH 7.2, one to 1.5 h at room temperature.

    Article Title: G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L
    Article Snippet: Antibodies Anti-ATG14L (MBL, PD026,Lot.003), anti-ZBTB16 (ab39354, abcam), anti-Beclin1 (sc-H-300, Santa Cruz), anti-Vps34 (12452-1-AP, PTG), anti LC3B (L7543, Sigma), anti-P62 (PM045, MBL), anti-P-GSK3β (ser9) (#9323, CST), anti-GSK3β (#9832S, CST), anti-p44/42 MAPK kinase (#9102, CST), anti-p-p44/42 MAPK (T202/Y204) (#9101S, CST), anti-pAKT (S473) (#4060S, CST), anti-AKT (sc-H136, Santa Cruz), anti-Tubulin (PM054, MBL), anti-Myc (M4439, Sigma), anti-Flag M2 (F1804, Sigma), anti-Cullin3 (ab75851, abcam), anti-Xpress (R910-25, Invitrogen), anti-phosphoserine (44911M, Invitrogen), anti-p-Threonine-proline (#9391S, CST), Gαq/11 antibody (C-19) (sc-392, Santa Cruz), Gαs antibody (A-16) (sc-26766, Santa Cruz), anti-Gα13 (sc-410, Santa Cruz), anti-Ubiquitin (Z0458, Dako), mouse EM48 (MAB5374, Millipore). .. Immunoprecipitation Cells were lysed with NP-40 buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitors cocktail [Sigma], 5% glycerol, 10 mM NaF, 1 mM PMSF), or Buffer II (1 mM EDTA, 0.1% NP-40, 10 mM Tris-HCl pH 7.5). .. Whole cell lysates obtained by centrifugation were incubated with 5 mg of antibody and protein G agarose beads (Invitrogen) overnight at 4°C.

    Centrifugation:

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: HEK293 cells in a 10-cm dish were transfected with a pShuttle-CMV expression vector for Flag-tagged hHSF1 or an hHSF1 point mutant, and were lysed with NP-40 lysis buffer. .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Western Blot:

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: HEK293 cells in a 10-cm dish were transfected with a pShuttle-CMV expression vector for Flag-tagged hHSF1 or an hHSF1 point mutant, and were lysed with NP-40 lysis buffer. .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

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    Millipore non ionic detergent triton x 114
    Triton X-114 solubility of different cell-associated and secreted PPAD species.  P .  gingivalis  isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure   S1 .
    Non Ionic Detergent Triton X 114, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non ionic detergent triton x 114/product/Millipore
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    97
    Millipore co immunoprecipitation buffer
    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 <t>co-immunoprecipitation</t> assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.
    Co Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/co immunoprecipitation buffer/product/Millipore
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    Triton X-114 solubility of different cell-associated and secreted PPAD species.  P .  gingivalis  isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure   S1 .

    Journal: Scientific Reports

    Article Title: Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis

    doi: 10.1038/s41598-018-27223-5

    Figure Lengend Snippet: Triton X-114 solubility of different cell-associated and secreted PPAD species. P . gingivalis isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure  S1 .

    Article Snippet: Aqueous two-phase system protein purification To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 (Sigma-Aldrich, St. Louis, USA) .

    Techniques: Solubility, Cell Culture, Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot, Marker

    A-LPS modification of PPAD in sorting type I and II isolates of  P .  gingivalis . Cells of  P .  gingivalis  sorting type I and II isolates were cultured in BHI and, subsequently, extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting. In panel A immunodetection was performed with an A-LPS-specific monoclonal antibody. Panel B shows a dual-labeling image upon immunodetection with the A-LPS-specific monoclonal antibody (green signal) and a PPAD-specific rabbit antibody (red signal). Bands representing the A-LPS-modified PPAD species of ~75–85-kDa PPAD are boxed in both panels. Names of sorting type I isolates are underlined. Molecular weights of marker proteins are indicated. The full-length blot is presented in Figure   S1 . Please note that the order of ‘A’- and ‘T’-labeled lanes is inversed compared to Fig.   2  as samples were loaded in a different order.

    Journal: Scientific Reports

    Article Title: Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis

    doi: 10.1038/s41598-018-27223-5

    Figure Lengend Snippet: A-LPS modification of PPAD in sorting type I and II isolates of P . gingivalis . Cells of P . gingivalis sorting type I and II isolates were cultured in BHI and, subsequently, extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting. In panel A immunodetection was performed with an A-LPS-specific monoclonal antibody. Panel B shows a dual-labeling image upon immunodetection with the A-LPS-specific monoclonal antibody (green signal) and a PPAD-specific rabbit antibody (red signal). Bands representing the A-LPS-modified PPAD species of ~75–85-kDa PPAD are boxed in both panels. Names of sorting type I isolates are underlined. Molecular weights of marker proteins are indicated. The full-length blot is presented in Figure  S1 . Please note that the order of ‘A’- and ‘T’-labeled lanes is inversed compared to Fig.  2 as samples were loaded in a different order.

    Article Snippet: Aqueous two-phase system protein purification To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 (Sigma-Aldrich, St. Louis, USA) .

    Techniques: Modification, Cell Culture, Polyacrylamide Gel Electrophoresis, Western Blot, Immunodetection, Labeling, Marker

    Hsp90 is required for TNF-triggered necroptosis. ( a ,  b ) 17AAG treatment inhibits TNF-induced necroptosis. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 24 h. Cell images were taken with a Nikon-TE2000 microscope ( a ). Scale bar, 200  μ m. Cell viability was determined by an MTS assay ( b ). Results are the means±S.D. of triplicate measurements. The final concentrations of 20 ng/ml TNF, 100 nM Smac mimetic, 20  μ M z-VAD and 10  μ M necrostatin-1 were used. T, TNF; S, Smac mimetic; Z, z-VAD; Nec-1, necrostatin-1. ( c ) 17AAG treatment reduces the phosphorylation of MLKL. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The activation of MLKL was analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. Protein levels were detected by western blotting using anti-RIP1, anti-RIP3, anti-MLKL and anti-actin antibodies. ( d ) 17AAG inhibits TNF-induced oligomerization of phosphorylated MLKL. HT29 cells were treated as in ( c ). The cells were harvested and lysed, and non-reducing samples (without  β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. ( e ) 17AAG inhibits plasma membrane translocation of phosphorylated MLKL. HT29 cells were pretreated with or without 125 nM 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The cells were harvested and then separated into the aqueous phase (Aq) and detergent phase (Det) using Triton X-114 lysis buffer as described in the experimental procedures. The samples were analyzed by western blotting with the indicated antibodies. ( f ) The effect of 17AAG on TNF-induced necrosome formation. Ten-centimetre plates of HT29 cells were pretreated with or without 125 nM 17AAG for 12 h and then stimulated with TNF alone or TSZ for 8 h. Cells were then harvested and whole-cell extracts were immunoprecipitated with anti-RIP3 antibody or anti-IgG antibody and subsequently analyzed by western blotting for the indicated proteins. The asterisks denote nonspecific IgG bands

    Journal: Cell Death & Disease

    Article Title: Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

    doi: 10.1038/cddis.2015.390

    Figure Lengend Snippet: Hsp90 is required for TNF-triggered necroptosis. ( a , b ) 17AAG treatment inhibits TNF-induced necroptosis. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 24 h. Cell images were taken with a Nikon-TE2000 microscope ( a ). Scale bar, 200  μ m. Cell viability was determined by an MTS assay ( b ). Results are the means±S.D. of triplicate measurements. The final concentrations of 20 ng/ml TNF, 100 nM Smac mimetic, 20  μ M z-VAD and 10  μ M necrostatin-1 were used. T, TNF; S, Smac mimetic; Z, z-VAD; Nec-1, necrostatin-1. ( c ) 17AAG treatment reduces the phosphorylation of MLKL. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The activation of MLKL was analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. Protein levels were detected by western blotting using anti-RIP1, anti-RIP3, anti-MLKL and anti-actin antibodies. ( d ) 17AAG inhibits TNF-induced oligomerization of phosphorylated MLKL. HT29 cells were treated as in ( c ). The cells were harvested and lysed, and non-reducing samples (without β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. ( e ) 17AAG inhibits plasma membrane translocation of phosphorylated MLKL. HT29 cells were pretreated with or without 125 nM 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The cells were harvested and then separated into the aqueous phase (Aq) and detergent phase (Det) using Triton X-114 lysis buffer as described in the experimental procedures. The samples were analyzed by western blotting with the indicated antibodies. ( f ) The effect of 17AAG on TNF-induced necrosome formation. Ten-centimetre plates of HT29 cells were pretreated with or without 125 nM 17AAG for 12 h and then stimulated with TNF alone or TSZ for 8 h. Cells were then harvested and whole-cell extracts were immunoprecipitated with anti-RIP3 antibody or anti-IgG antibody and subsequently analyzed by western blotting for the indicated proteins. The asterisks denote nonspecific IgG bands

    Article Snippet: The following reagents were used: Hsp90 inhibitor 17AAG (Selleckchem, Huston, TX, USA, S1141); z-VAD-fmk (ApexBio, Huston, TX, USA, A1902); Triton X-114 (Sigma-Aldrich, St. Louis, MO, USA); anti-HA (sc-805), anti-Myc (sc-40), anti-RIP3 (sc-374639) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Hsp90 (ab178854), anti-phospho-MLKL (ab187091) (Abcam, Cambridge, MA, USA); anti-MLKL (Merck Millipore, Billerica, MA, USA, MABC604); anti-Flag M2 (Sigma-Aldrich); anti-RIP1 (BD Pharmingen, Franklin Lakes, NJ, USA, 51-6559GR).

    Techniques: Microscopy, MTS Assay, Activation Assay, Western Blot, Translocation Assay, Lysis, Immunoprecipitation

    Coexpression of Hsp90 enhances MLKL-mediated necroptosis. ( a ) The 293T cells were transfected with vector, with Flag-Hsp90 α , with MLKL-Myc alone, or with Flag-Hsp90 α  and MLKL-Myc. After 24 h of transfection, cell images were taken with a Nikon-TE2000 microscope. Scale bar, 100  μ m. ( b ) The 293T cells were transfected as in ( a ). After 24 h of transfection, cell death was quantified by propidium iodide (PI) staining. Cell death data are the means±S.D. of three independent experiments. ( c ) Hsp90 α  increases MLKL oligomerization. The 293T cells were transfected with the indicated plasmids. The cells were harvested 24 h after transfection, and non-reducing samples (without  β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with anti-HA antibody. ( d ) Hsp90 α  increases the plasma membrane translocation of MLKL. The 293T cells were transfected with the indicated expression vectors. After 24 h of transfection, the cells were harvested and lysed in Triton X-114 lysis buffer and then separated into aqueous phase (Aq) and detergent phase (Det) as described in the experimental procedures. The samples were resolved and probed with the indicated antibodies.  β -actin and Cox4 were used as loading controls for soluble protein and membrane protein, respectively

    Journal: Cell Death & Disease

    Article Title: Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

    doi: 10.1038/cddis.2015.390

    Figure Lengend Snippet: Coexpression of Hsp90 enhances MLKL-mediated necroptosis. ( a ) The 293T cells were transfected with vector, with Flag-Hsp90 α , with MLKL-Myc alone, or with Flag-Hsp90 α and MLKL-Myc. After 24 h of transfection, cell images were taken with a Nikon-TE2000 microscope. Scale bar, 100  μ m. ( b ) The 293T cells were transfected as in ( a ). After 24 h of transfection, cell death was quantified by propidium iodide (PI) staining. Cell death data are the means±S.D. of three independent experiments. ( c ) Hsp90 α increases MLKL oligomerization. The 293T cells were transfected with the indicated plasmids. The cells were harvested 24 h after transfection, and non-reducing samples (without β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with anti-HA antibody. ( d ) Hsp90 α increases the plasma membrane translocation of MLKL. The 293T cells were transfected with the indicated expression vectors. After 24 h of transfection, the cells were harvested and lysed in Triton X-114 lysis buffer and then separated into aqueous phase (Aq) and detergent phase (Det) as described in the experimental procedures. The samples were resolved and probed with the indicated antibodies. β -actin and Cox4 were used as loading controls for soluble protein and membrane protein, respectively

    Article Snippet: The following reagents were used: Hsp90 inhibitor 17AAG (Selleckchem, Huston, TX, USA, S1141); z-VAD-fmk (ApexBio, Huston, TX, USA, A1902); Triton X-114 (Sigma-Aldrich, St. Louis, MO, USA); anti-HA (sc-805), anti-Myc (sc-40), anti-RIP3 (sc-374639) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Hsp90 (ab178854), anti-phospho-MLKL (ab187091) (Abcam, Cambridge, MA, USA); anti-MLKL (Merck Millipore, Billerica, MA, USA, MABC604); anti-Flag M2 (Sigma-Aldrich); anti-RIP1 (BD Pharmingen, Franklin Lakes, NJ, USA, 51-6559GR).

    Techniques: Transfection, Plasmid Preparation, Microscopy, Staining, Translocation Assay, Expressing, Lysis

    CopB and CopB2 partition differently in Triton X-114 extracts. Integral membrane association was tested by extracting  C. trachomatis  L2-infected HeLa cultures with Triton X-114 and separating proteins into aqueous and detergent soluble fractions. (A) Cultures were harvested at 20 h postinfection, and a parallel, mock-treated HeLa culture was harvested as a specificity control. Equivalent quantities of total protein from aqueous (Aq)- and detergent (Det)-soluble samples were resolved by SDS-PAGE in 12% polyacrylamide gels. Immunoblots were probed with anti-MOMP, anti-GroEL, anti-CopN, or anti-IncG as a fractionation control and with antibody specific for CopB or CopB2. Proteins were visualized by probing with secondary antibodies conjugated to alkaline phosphatase and development with NBT-BCIP. (B) HeLa cultures were mock treated or  C. trachomatis  L2 infected at an MOI of 10. Cultures were harvested at 2 h postinfection, extracted with Triton X-114, and probed by immunoblotting with the indicated antibodies. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

    Journal: Infection and Immunity

    Article Title: Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions ▿

    doi: 10.1128/IAI.00159-11

    Figure Lengend Snippet: CopB and CopB2 partition differently in Triton X-114 extracts. Integral membrane association was tested by extracting C. trachomatis L2-infected HeLa cultures with Triton X-114 and separating proteins into aqueous and detergent soluble fractions. (A) Cultures were harvested at 20 h postinfection, and a parallel, mock-treated HeLa culture was harvested as a specificity control. Equivalent quantities of total protein from aqueous (Aq)- and detergent (Det)-soluble samples were resolved by SDS-PAGE in 12% polyacrylamide gels. Immunoblots were probed with anti-MOMP, anti-GroEL, anti-CopN, or anti-IncG as a fractionation control and with antibody specific for CopB or CopB2. Proteins were visualized by probing with secondary antibodies conjugated to alkaline phosphatase and development with NBT-BCIP. (B) HeLa cultures were mock treated or C. trachomatis L2 infected at an MOI of 10. Cultures were harvested at 2 h postinfection, extracted with Triton X-114, and probed by immunoblotting with the indicated antibodies. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

    Article Snippet: For Triton X-114 extractions, soluble and integral membrane proteins were separated based on solubility in Triton X-114 (Sigma) to examine the partitioning of selected proteins during chlamydial infection.

    Techniques: Infection, SDS Page, Western Blot, Fractionation

    Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: Patch 1 and Patch 2 residues of the AP2-μ2 subunit that bind to PIP 2 lipid are not critical for the binding of the AP2 complex to CXCR2 A) Non-silencing (NS), HEK-293-μ2-KD cells and HEK-293-μ2-KD cells with transiently over-expressed AP2-μ2 mutants (P1, P1+P2, P1P2T and WT) were serum starved, stimulated with 100 ng /ml CXCL8 and cross-linked with DSP. The cells were lysed and a CXCR2 co-immunoprecipitation assay was performed. The CXCR2 associated proteins were eluted with 50 mM DTT and separated by 10%SDS-PAGE. The CXCR2 associated AP2 complex was probed with an anti-β2 antibody. Experiments were repeated 3 times and the mean band densities as normalized to co-immunoprecipitation with CXCR2 ±S. D. are shown. B) HA-AP2-μ2 associates with endogenous α2 subunit of AP-2. HEK-293-μ2-KD cells with transiently over-expressed HA-AP2-μ2 mutants (P1, P1+P2, T, P1P2T and WT), were lysed subjected to Western blot analysis for α2 and HA-μ2 subunits (upper panel). Each HA-tagged μ2 subunit was immunoprecipitated with anti-HA-agarose and blotted for endogenous α2 and for HA-μ2 (upper panel). A representative blot from 3 individual experiments is shown. C) Functional AP-2 complexes successfully incorporate transiently expressed HA-AP2-μ2 as shown by a reciprocal co-immunoprecipitation. A reciprocal co-immunoprecipitation of the endogenous AP2-β2 from 1.5 mg of total lysate shows that the functional AP-2 complexes contain both endogenous AP2-α2 and overexpressed HA-AP2-μ2. One thirtieth (1/30) of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. The top panel shows co-immunoprecipitation and the bottom panel shows the lysates. A representative blot from 2 individual experiments is shown.

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Immunoprecipitation, Western Blot, Functional Assay

    AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Adaptor Protein2 (AP2) orchestrates CXCR2-mediated cell migration

    doi: 10.1111/tra.12154

    Figure Lengend Snippet: AP2 is essential for CXCR2-mediated chemotaxis, but β-arrestin1 is dispensable A) Top panel: LLKIL motif in CTDs of human CXC chemokine receptors is conserved. The CTDs of CXCR2 (45 residues), CXCR1 (44 residues), CXCR3 (49 residues) and CXCR4 (47 residues) were aligned with CLUSTALW (1.83) multiple sequence alignment program. The LLKIL functional motif of CXCR2 and similar putative motifs in other CXC receptor CTDs are in bold. Also, the serine residues known to be phosphorylated in CXCR2 CTD in response to CXCL8 stimulation are in bold. Bottom panel: The mutations in CXCR2 important for binding of AP2 and β-arrestin are illustrated. B) Decreased association of CXCR2 mutants with AP2 and/or β-arrestin1 after stimulation with CXCL8. dHL-60 cells stably expressing CXCR2-WT, 4A or CXCR2-4A/IL mutants were stimulated with or without CXCL8. CXCR2 was immunoprecipitated with anti-CXCR2 antibody, and blotted for AP2-β2 subunit or β-arrestin1. The blot was stripped and re-blotted for CXCR2. The relative values of fold increase in response to CXCL8 stimulation for each cell line calculated from 3 independent experiments is shown under the western blots (fold ± S.E.M.). One tenth of the total lysate input for co-immunoprecipitation was used for Western analysis of the total lysates. C) CXCL8-mediated internalization of CXCR2 is abolished in 4A/IL mutant of CXCR2, but only partially attenuated in 4A-CXCR2 mutant. The internalization of CXCR2 was performed by following the internalization of 125 I-CXCL8 in dHL60-CXCR2 cells stably expressing CXCR2-WT, 4A or 4A/IL mutant. Error bars are S.E.M and the experiments were repeated 3 times with duplicates for each treatment. ANOVA: 2 min – 4A vs. 4A/IL, p

    Article Snippet: The cells were lysed in ice-cold co-immunoprecipitation buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA) containing proteinase inhibitor cocktail I and phosphatase inhibitor cocktails 3 and 2 (Sigma/Aldrich, St. Louis, MO).

    Techniques: Chemotaxis Assay, Sequencing, Functional Assay, Binding Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis