2 mercaptoethanol Millipore Search Results


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  • 99
    Thermo Fisher β mercaptoethanol
    ER-retention of APP by addition of the di-lysine motif KKAA induces SDS stable homo-dimers in an oxidizing cellular surrounding. a Schematic illustration of the APP domain structure and the used dilysine retention constructs. APP isoform 695 (APP695) was fused to a C-terminal myc-tag, followed by either a KKAA ER retention motif, or a “leaky” KKFF motif allowing trafficking to the cis -Golgi compartment. The E1 domain, acidic domain, E2 domain, transmembrane domain (TM), and the intracellular domain are indicated. b CHO-K1 cells stably over expressing the respective APP695 wt or APP695 retention constructs were subjected to cell surface biotinylation. To inhibit endocytosis from the cell surface, AP180 D/N (lane four) was transiently co-expressed. At 24-h post-transfection, cell surface proteins were labeled with sulfo NHS-SS-biotin at 4°C, prior to lysis. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and Western blot (PD: NeutrAvidin; right panel ). As input controls, 20 μg of cell lysates were used (input; left panel ). APP full length, CTFs and AP180 D/N were probed with 9E10 antibody. Monoclonal α-Tubulin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. For the input control, all samples were treated with SDS sample loading buffer, containing <t>β-mercaptoethanol</t> (BME) without heat denaturing prior to SDS gel electrophoresis. To elute surface proteins from NeutrAvidin beads, samples were denatured for 5 min at 95°C with BME and separated on 4–12% SDS gels. Molecular weight standards (kDa) are indicated on the left . c Cell lysates (20 μg each lane) of CHO-K1 cells stably overexpressing APP ER (KKAA), were incubated with SDS sample loading buffer (+ BME) and heat denatured for 10 min at the indicated temperatures (4–85°C). APP monomers and SDS stable dimers were detected, using the monoclonal anti myc antibody (9E10), shown on one representative Western blot. d APP dimers and monomers were quantified by densitometric analysis of Western blots, as the one shown in c . In each case, intensities at 4°C were set as 100%. Diagram shows relative intensity (%) of APP monomers and dimers, plotted against different temperatures. Data are ( n ≥ 3) ± SEM, one-way ANOVA
    β Mercaptoethanol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 2 mercaptoethanol
    GSH, but not GSSG or other antioxidants, inhibits ceramide generation induced by H 2 O 2 in lung epithelial cells. A549 cells were preincubated for 1 h in medium supplemented with 1% serum in the presence or absence of 10 mM of GSH, GSSG, DTT, or <t>2-mercaptoethanol,</t> followed by incubations with 250 μM H 2 O 2 for an additional 30 min. The cells were rinsed with ice-cold PBS to terminate the treatments and harvested by trypsinization. GSH (A) and ceramide (B) levels were determined as described in Materials and Methods. Values represent mean ± SEM. *Mean of the group that was significantly different from the mean of the H 2 O 2 -treated group ( P
    2 Mercaptoethanol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 22735 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glutamine
    GSH, but not GSSG or other antioxidants, inhibits ceramide generation induced by H 2 O 2 in lung epithelial cells. A549 cells were preincubated for 1 h in medium supplemented with 1% serum in the presence or absence of 10 mM of GSH, GSSG, DTT, or <t>2-mercaptoethanol,</t> followed by incubations with 250 μM H 2 O 2 for an additional 30 min. The cells were rinsed with ice-cold PBS to terminate the treatments and harvested by trypsinization. GSH (A) and ceramide (B) levels were determined as described in Materials and Methods. Values represent mean ± SEM. *Mean of the group that was significantly different from the mean of the H 2 O 2 -treated group ( P
    Glutamine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore immobilon p membranes
    T β R-II protein expression in total lysate, cytosol, and membrane fractions from human breast cell lines. Aliquots (10 μg) of total cell lysate and fractionated protein were analyzed by SDS-PAGE electrophoresis, transferred to <t>PVDF</t> membranes, blocked with 5% nonfat dry milk in PBS-T, and incubated with T β R-II antibody. Bound antibodies were detected by incubation with peroxidase-conjugated antirabbit immunoglobulin and enhanced chemiluminscent autoradiography. A 70-kDa band corresponding to the size expected for T β R-II protein was observed in all cell lines in total lysate and the cytosolic fraction. The membrane fractions demonstrate the clear presence of T β R-II protein in the Mv1Lu positive control, the MCF-10F nontumorigenic, and the tumorigenic MDA-MB231 cell lines. The other cell lines have little or no T β R-II protein present in the membrane fraction.
    Immobilon P Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16064 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore hepes
    Interactions between filamentous actin, GFP-Lpd (850–1250aa), and GFP-LZ-Lpd (850–1250aa) measured by cosedimentation at different buffer ionic strengths. ( A ) Monomeric GFP-Lpd 850−1250aa and dimeric GFP-LZ-Lpd 850−1250aa interact with filamentous actin in the presence of 50, 100, 150 mM <t>KCl.</t> SDS-PAGE from three experiments showing the cosedimentation of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa (monomeric protein concentration). Buffer composition is 20 mM <t>HEPES</t> [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl 2 , 0.5 mM EGTA. ( B ) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). ( C ) SDS-PAGE as in Figure 1H , showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). ( D ) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl 2 , and 0.5 mM EGTA. ( C , D ) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification. DOI: http://dx.doi.org/10.7554/eLife.06585.004
    Hepes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50050 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sodium pyruvate
    Interactions between filamentous actin, GFP-Lpd (850–1250aa), and GFP-LZ-Lpd (850–1250aa) measured by cosedimentation at different buffer ionic strengths. ( A ) Monomeric GFP-Lpd 850−1250aa and dimeric GFP-LZ-Lpd 850−1250aa interact with filamentous actin in the presence of 50, 100, 150 mM <t>KCl.</t> SDS-PAGE from three experiments showing the cosedimentation of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa (monomeric protein concentration). Buffer composition is 20 mM <t>HEPES</t> [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl 2 , 0.5 mM EGTA. ( B ) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). ( C ) SDS-PAGE as in Figure 1H , showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). ( D ) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl 2 , and 0.5 mM EGTA. ( C , D ) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification. DOI: http://dx.doi.org/10.7554/eLife.06585.004
    Sodium Pyruvate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore protease inhibitor cocktail
    Interactions between filamentous actin, GFP-Lpd (850–1250aa), and GFP-LZ-Lpd (850–1250aa) measured by cosedimentation at different buffer ionic strengths. ( A ) Monomeric GFP-Lpd 850−1250aa and dimeric GFP-LZ-Lpd 850−1250aa interact with filamentous actin in the presence of 50, 100, 150 mM <t>KCl.</t> SDS-PAGE from three experiments showing the cosedimentation of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa (monomeric protein concentration). Buffer composition is 20 mM <t>HEPES</t> [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl 2 , 0.5 mM EGTA. ( B ) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). ( C ) SDS-PAGE as in Figure 1H , showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). ( D ) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl 2 , and 0.5 mM EGTA. ( C , D ) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification. DOI: http://dx.doi.org/10.7554/eLife.06585.004
    Protease Inhibitor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 134786 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore edta
    ITC results for binding of (A) WT RelA-NLS(293-321) (B) F309A RelA-NLS(293-321). (C) WT RelA(190-321)/p50(248-350) (D) F309A RelA(190-321)/p50(248-350) to IκBα(67-206) in 50 mM NaCl, 25 mM <t>Tris,</t> pH 7.5, 0.5mM <t>EDTA,</t> and 0.5mM sodium azide
    Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore penicillin streptomycin
    ITC results for binding of (A) WT RelA-NLS(293-321) (B) F309A RelA-NLS(293-321). (C) WT RelA(190-321)/p50(248-350) (D) F309A RelA(190-321)/p50(248-350) to IκBα(67-206) in 50 mM NaCl, 25 mM <t>Tris,</t> pH 7.5, 0.5mM <t>EDTA,</t> and 0.5mM sodium azide
    Penicillin Streptomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore triton x 100
    Presence of p14 in plasma membrane and soluble part of acrosome. ( A ) Western blot analysis showing the expression of p14 in membrane and cytosolic fractions of sperm and whole cell lysate. ( B ) Expression of the p14 in the cytosolic fraction of epididymis and cauda epididymal plasma. ( C ) Western blot showing the presence of p14 in plasma membranes and soluble components of the acrosome, acrosomal matrix and sperm cell lysate. ( D ) Immunoblot with a peripheral plasma membrane protein extract prepared from washed caudal epididymis following treatment with AES solution and cell lysate. Proteins were extracted from caudal sperm with 0.625% <t>Triton-X-100</t> solution for immunoblotting (details in methods ). Experiments were repeated four times.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher l glutamine
    Presence of p14 in plasma membrane and soluble part of acrosome. ( A ) Western blot analysis showing the expression of p14 in membrane and cytosolic fractions of sperm and whole cell lysate. ( B ) Expression of the p14 in the cytosolic fraction of epididymis and cauda epididymal plasma. ( C ) Western blot showing the presence of p14 in plasma membranes and soluble components of the acrosome, acrosomal matrix and sperm cell lysate. ( D ) Immunoblot with a peripheral plasma membrane protein extract prepared from washed caudal epididymis following treatment with AES solution and cell lysate. Proteins were extracted from caudal sperm with 0.625% <t>Triton-X-100</t> solution for immunoblotting (details in methods ). Experiments were repeated four times.
    L Glutamine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 146399 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fbs
    STAP-2 enhances proliferation of <t>DU145</t> cells in vitro and in vivo . A , qPCR analysis for mRNA levels of STAP-2 in various cancer cell lines and human tissues. B and C , expression levels of STAP-2 in STAP-2–knockdown DU145 cells were determined by qPCR ( B ) and immunoblotting ( IB ) ( C ). TCL , total cell lysates. D , viable cells of control or STAP-2 shRNA–expressing DU145 cells were measured by a WST assay. E , control or STAP-2 shRNA–expressing DU145 cells were cultured in DMEM containing 1% <t>FBS</t> for the indicated times, and then the surviving cells were measured by a WST assay. F , STAP-2–knockdown DU145 cells were transfected with pcDNA3.1-Myc-STAP-2, and then their cell proliferation was measured by a WST assay at 72 h post-transfection, as in D . An aliquot of total cell lysates was immunoblotted by anti-Myc and anti-actin antibodies. G , expression levels of STAP-2 in STAP-2–knockdown LNCaP cells were determined by immunoblotting. H , viable cells of control or STAP-2 shRNA–expressing LNCaP cells were measured by a WST assay. I , DU145 cells (5.0 × 10 6 cells) were subcutaneously injected into BALB/c nude mice (day 0), and tumor volume was measured. Tumors were established in 5 of 5 mice (shControl) and 3 of 5 mice (shSTAP-2). J , weight of tumors and spleens from the tumor-bearing mice at day 33. n = 3; mean values and S.E. ( error bars ) are depicted. *, p
    Fbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore leukemia inhibitory factor
    Analysis of 5-mC and 5-hmC during embryonic stem cell differentiation to embryoid body. A , <t>LIF</t> withdrawal leading to embryonic stem cell marker Oct4 and Nanog repression is shown. Shown is a Western blot of ES cell extract after LIF withdrawal along with NIH3T3 (3T3), as shown on the top , with the indicated antibodies on the left . Gapdh is the loading control. B , Oct4 and Nanog gene are methylated upon embryoid body formation. Day post-LIF withdrawal is on the left along with NIH3T3. UDP-Glc and <t>β-GT</t> are shown on the top along with restriction enzyme MspI ( M ) and HpaII ( H ) cleavage along with control ( C ). C , 5-hmC detection during embryoid body formation is shown. Day post-LIF withdrawal is shown on the left. D , global methylation analysis using LC-MS during embryoid body formation is shown. Each analysis point was performed in duplicate. Deoxycytosine (C), 5-methyldeoxycytosine (5-mC or mC ) and 5-hydroxymethyldeoxycytosine (5-hmC or hmC ) are shown.
    Leukemia Inhibitory Factor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5533 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rpmi 1640
    sPLA 2 s induce the release of VEGF-A and VEGF-C from HLMs. A and B , HLMs were incubated (37°C, 24 h) with <t>RPMI</t> 1640 alone (Control) or with the indicated concentrations of hGIIA, hGX, or LPS. VEGF-A ( A ) and VEGF-C ( B ) release was determined by ELISA. Data are mean ± SEM of four experiments. * p
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    Millipore β mercaptoethanol
    In vitro copper reductase assay with the Xenopus laevis H3-H4 tetramer. (A) Standard curve for Cu 1+ detection for copper reductase assay conditions used with X. laevis tetramers with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:10 in presence of 2 mM <t>β-Mercaptoethanol</t> as reducing agent (see Methods). Error bars indicate mean ± SD of three replicate measurements. (B) Same as (A) but for reactions with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:15 (see Methods). (C) Progress curve of copper reduction using 30 µM TCEP as the electron donor with 1 µM of X. laevis tetramer, equimolar monomeric X. laevis histone H3, and equimolar monomeric S. cerevisiae histone H2A. (D) Henri-Michaelis-Menten curves of initial velocities of reactions with the X. laevis tetramer. (E) Calculated enzymatic parameters of the tetramer from the curve in (D). (F) Same as (C) but with X. laevis tetramer, or equimolar RNase A or DTT. (G) Same as (A) but for reactions with increasing amounts of CuCl 2 -ADA pH 8 at a ratio 1:1 (see Methods). (H) Same as (A) but for reactions with increasing amounts of CuCl 2 -NTA pH 7.6 at a ratio 1:1 (see Methods). (I) Progress curves of copper reduction by 5 µM of the indicated X. laevis tetramers in presence of 200 µM TCEP and 0.5 mM CuCl 2 in 0.5 mM NTA pH 7.6.
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    Millipore mgcl2
    PPM1H dephosphorylates Rab10 in vitro. ( A ) The indicated amounts of recombinant wild-type and mutant PPM1H (with a His-Sumo N-terminal tag, expressed in E. coli ) were incubated in vitro with 2.5 µg pT73 ~60% phosphorylated Rab10[1–181, GDP bound] for 30 min in the presence of 10 mM <t>MgCl2</t> in 40 mM HEPES pH 7.0 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by phos-tag gel electrophoresis that separates phosphorylated and dephosphorylated Rab10. Gel was stained with Instant Blue Coomassie. Bands corresponding to phosphorylated and non-phosphorylated Rab10 are marked with open (○) and closed (●) circles respectively. ( B ) As in ( A ) except that a time-course assay was performed using 2.5 µg pT73 phosphorylated Rab10[1–181, GDP bound] and 40 ng wild-type or mutant PPM1H for the indicated times. ( C ) As in ( A ) except that PPM1J was assessed. ( D ) As in ( A ) except and PPM1M was assessed.
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    Millipore rpmi 1640 medium
    LPS of B . pseudomallei signals solely via murine TLR4. Murine whole blood (A) and peritoneal macrophages (B) harvested from wild-type (WT), TLR2 -/- and TLR4 -/- mice were stimulated for 24h with <t>RPMI</t> 1640 + 10% FCS medium, LPS of E . coli 0111:B4 (100 ng/ml), LPS of B . pseudomallei (100 ng/ml), heat-killed B . pseudomallei 1026b (100 ng/ml), or its O-antigen lacking mutant SRM117 (both 10 7 CFU/ml) before measurement of murine TNF-α (n = 4). Following a Kruskal-Wallis test, Mann-Whitney U-tests were performed. Data are means ± SEM. Results are representative of two or three independent experiments. * P
    Rpmi 1640 Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 30869 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tris
    2D 1 H- 15 N TROSY spectrum of 0.2mM selectively 15 N-isoleucine-labeled arrestin-1 in 25 mM <t>Bis-Tris,</t> 150mM <t>NaCl</t> and 5mM mercaptoethanol, pH=6.5 at 308 K using a Bruker Avance 800MHz spectrometer. 11 out of 20 isoleucine residue peaks were assigned, as labeled
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    Millipore lps
    K45 of RIPK1 promotes necroptotic signaling. (a, b , e – h ) WT or RIPK1 K45A macrophages were treated as indicated in figure panels with <t>LPS</t> (100 ng/ml) and <t>TNF</t> α (50 ng/ml) with or without zVAD-fmk (50 μ M), and samples were evaluated by western blotting. Panels ( a ) and ( b ) shows western blotting at 3 h posttreatment. Panel ( h ) shows western blotting at 4 h posttreatment. ( c , d , i – k ) Densitometry of pooled western blottings from several experiments. ANOVA with Bonferroni post-test was used to measure statistical significance, * P
    Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lysis buffer
    K45 of RIPK1 promotes necroptotic signaling. (a, b , e – h ) WT or RIPK1 K45A macrophages were treated as indicated in figure panels with <t>LPS</t> (100 ng/ml) and <t>TNF</t> α (50 ng/ml) with or without zVAD-fmk (50 μ M), and samples were evaluated by western blotting. Panels ( a ) and ( b ) shows western blotting at 3 h posttreatment. Panel ( h ) shows western blotting at 4 h posttreatment. ( c , d , i – k ) Densitometry of pooled western blottings from several experiments. ANOVA with Bonferroni post-test was used to measure statistical significance, * P
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    K45 of RIPK1 promotes necroptotic signaling. (a, b , e – h ) WT or RIPK1 K45A macrophages were treated as indicated in figure panels with <t>LPS</t> (100 ng/ml) and <t>TNF</t> α (50 ng/ml) with or without zVAD-fmk (50 μ M), and samples were evaluated by western blotting. Panels ( a ) and ( b ) shows western blotting at 3 h posttreatment. Panel ( h ) shows western blotting at 4 h posttreatment. ( c , d , i – k ) Densitometry of pooled western blottings from several experiments. ANOVA with Bonferroni post-test was used to measure statistical significance, * P
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    K45 of RIPK1 promotes necroptotic signaling. (a, b , e – h ) WT or RIPK1 K45A macrophages were treated as indicated in figure panels with <t>LPS</t> (100 ng/ml) and <t>TNF</t> α (50 ng/ml) with or without zVAD-fmk (50 μ M), and samples were evaluated by western blotting. Panels ( a ) and ( b ) shows western blotting at 3 h posttreatment. Panel ( h ) shows western blotting at 4 h posttreatment. ( c , d , i – k ) Densitometry of pooled western blottings from several experiments. ANOVA with Bonferroni post-test was used to measure statistical significance, * P
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    K45 of RIPK1 promotes necroptotic signaling. (a, b , e – h ) WT or RIPK1 K45A macrophages were treated as indicated in figure panels with <t>LPS</t> (100 ng/ml) and <t>TNF</t> α (50 ng/ml) with or without zVAD-fmk (50 μ M), and samples were evaluated by western blotting. Panels ( a ) and ( b ) shows western blotting at 3 h posttreatment. Panel ( h ) shows western blotting at 4 h posttreatment. ( c , d , i – k ) Densitometry of pooled western blottings from several experiments. ANOVA with Bonferroni post-test was used to measure statistical significance, * P
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    Functionalization procedure of PLLA/PLLA-PEG-NH 2 nanofibers (brown). Step 1: Amino groups are activated by exposing nanofibers to saturated water vapor. Step 2: Carboxyl groups are activated by hydrolysis. Blue represents the loaded drug; red is rhodamine <t>BSA;</t> and green is FITC-BSA. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein <t>isothiocyanate;</t> PEG, poly(ethylene glycol); PLLA, poly(L-lactide).
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    Image Search Results


    ER-retention of APP by addition of the di-lysine motif KKAA induces SDS stable homo-dimers in an oxidizing cellular surrounding. a Schematic illustration of the APP domain structure and the used dilysine retention constructs. APP isoform 695 (APP695) was fused to a C-terminal myc-tag, followed by either a KKAA ER retention motif, or a “leaky” KKFF motif allowing trafficking to the cis -Golgi compartment. The E1 domain, acidic domain, E2 domain, transmembrane domain (TM), and the intracellular domain are indicated. b CHO-K1 cells stably over expressing the respective APP695 wt or APP695 retention constructs were subjected to cell surface biotinylation. To inhibit endocytosis from the cell surface, AP180 D/N (lane four) was transiently co-expressed. At 24-h post-transfection, cell surface proteins were labeled with sulfo NHS-SS-biotin at 4°C, prior to lysis. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and Western blot (PD: NeutrAvidin; right panel ). As input controls, 20 μg of cell lysates were used (input; left panel ). APP full length, CTFs and AP180 D/N were probed with 9E10 antibody. Monoclonal α-Tubulin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. For the input control, all samples were treated with SDS sample loading buffer, containing β-mercaptoethanol (BME) without heat denaturing prior to SDS gel electrophoresis. To elute surface proteins from NeutrAvidin beads, samples were denatured for 5 min at 95°C with BME and separated on 4–12% SDS gels. Molecular weight standards (kDa) are indicated on the left . c Cell lysates (20 μg each lane) of CHO-K1 cells stably overexpressing APP ER (KKAA), were incubated with SDS sample loading buffer (+ BME) and heat denatured for 10 min at the indicated temperatures (4–85°C). APP monomers and SDS stable dimers were detected, using the monoclonal anti myc antibody (9E10), shown on one representative Western blot. d APP dimers and monomers were quantified by densitometric analysis of Western blots, as the one shown in c . In each case, intensities at 4°C were set as 100%. Diagram shows relative intensity (%) of APP monomers and dimers, plotted against different temperatures. Data are ( n ≥ 3) ± SEM, one-way ANOVA

    Journal: Cellular and Molecular Life Sciences

    Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

    doi: 10.1007/s00018-011-0882-4

    Figure Lengend Snippet: ER-retention of APP by addition of the di-lysine motif KKAA induces SDS stable homo-dimers in an oxidizing cellular surrounding. a Schematic illustration of the APP domain structure and the used dilysine retention constructs. APP isoform 695 (APP695) was fused to a C-terminal myc-tag, followed by either a KKAA ER retention motif, or a “leaky” KKFF motif allowing trafficking to the cis -Golgi compartment. The E1 domain, acidic domain, E2 domain, transmembrane domain (TM), and the intracellular domain are indicated. b CHO-K1 cells stably over expressing the respective APP695 wt or APP695 retention constructs were subjected to cell surface biotinylation. To inhibit endocytosis from the cell surface, AP180 D/N (lane four) was transiently co-expressed. At 24-h post-transfection, cell surface proteins were labeled with sulfo NHS-SS-biotin at 4°C, prior to lysis. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and Western blot (PD: NeutrAvidin; right panel ). As input controls, 20 μg of cell lysates were used (input; left panel ). APP full length, CTFs and AP180 D/N were probed with 9E10 antibody. Monoclonal α-Tubulin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. For the input control, all samples were treated with SDS sample loading buffer, containing β-mercaptoethanol (BME) without heat denaturing prior to SDS gel electrophoresis. To elute surface proteins from NeutrAvidin beads, samples were denatured for 5 min at 95°C with BME and separated on 4–12% SDS gels. Molecular weight standards (kDa) are indicated on the left . c Cell lysates (20 μg each lane) of CHO-K1 cells stably overexpressing APP ER (KKAA), were incubated with SDS sample loading buffer (+ BME) and heat denatured for 10 min at the indicated temperatures (4–85°C). APP monomers and SDS stable dimers were detected, using the monoclonal anti myc antibody (9E10), shown on one representative Western blot. d APP dimers and monomers were quantified by densitometric analysis of Western blots, as the one shown in c . In each case, intensities at 4°C were set as 100%. Diagram shows relative intensity (%) of APP monomers and dimers, plotted against different temperatures. Data are ( n ≥ 3) ± SEM, one-way ANOVA

    Article Snippet: Alternatively, samples were incubated with sample buffer without β-mercaptoethanol and heat denatured for 10 min. Proteins were electrophoresed on 4–12% NuPage (Novex®, Invitrogen) gradient gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA).

    Techniques: Construct, Stable Transfection, Expressing, Transfection, Labeling, Lysis, SDS Page, Western Blot, SDS-Gel, Electrophoresis, Molecular Weight, Incubation

    APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins

    Journal: Cellular and Molecular Life Sciences

    Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

    doi: 10.1007/s00018-011-0882-4

    Figure Lengend Snippet: APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins

    Article Snippet: Alternatively, samples were incubated with sample buffer without β-mercaptoethanol and heat denatured for 10 min. Proteins were electrophoresed on 4–12% NuPage (Novex®, Invitrogen) gradient gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA).

    Techniques: Transfection, Marker, Incubation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Staining, Expressing, Western Blot, Stable Transfection, Blocking Assay

    GSH, but not GSSG or other antioxidants, inhibits ceramide generation induced by H 2 O 2 in lung epithelial cells. A549 cells were preincubated for 1 h in medium supplemented with 1% serum in the presence or absence of 10 mM of GSH, GSSG, DTT, or 2-mercaptoethanol, followed by incubations with 250 μM H 2 O 2 for an additional 30 min. The cells were rinsed with ice-cold PBS to terminate the treatments and harvested by trypsinization. GSH (A) and ceramide (B) levels were determined as described in Materials and Methods. Values represent mean ± SEM. *Mean of the group that was significantly different from the mean of the H 2 O 2 -treated group ( P

    Journal: American journal of respiratory cell and molecular biology

    Article Title: Ceramide-Mediated Apoptosis in Lung Epithelial Cells Is Regulated by Glutathione

    doi: 10.1165/ajrcmb.25.6.4321

    Figure Lengend Snippet: GSH, but not GSSG or other antioxidants, inhibits ceramide generation induced by H 2 O 2 in lung epithelial cells. A549 cells were preincubated for 1 h in medium supplemented with 1% serum in the presence or absence of 10 mM of GSH, GSSG, DTT, or 2-mercaptoethanol, followed by incubations with 250 μM H 2 O 2 for an additional 30 min. The cells were rinsed with ice-cold PBS to terminate the treatments and harvested by trypsinization. GSH (A) and ceramide (B) levels were determined as described in Materials and Methods. Values represent mean ± SEM. *Mean of the group that was significantly different from the mean of the H 2 O 2 -treated group ( P

    Article Snippet: Bis-benzimide (Hoechst 33258), L-D-buthionine sulfoximine (BSO), aminotriazole (ATZ), GSH, GSSG, NAC, dithiothreitol (DTT), 2-mercaptoethanol, and all chemical reagents were from Sigma Chemical Co. (St. Louis, MO).

    Techniques:

    T β R-II protein expression in total lysate, cytosol, and membrane fractions from human breast cell lines. Aliquots (10 μg) of total cell lysate and fractionated protein were analyzed by SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% nonfat dry milk in PBS-T, and incubated with T β R-II antibody. Bound antibodies were detected by incubation with peroxidase-conjugated antirabbit immunoglobulin and enhanced chemiluminscent autoradiography. A 70-kDa band corresponding to the size expected for T β R-II protein was observed in all cell lines in total lysate and the cytosolic fraction. The membrane fractions demonstrate the clear presence of T β R-II protein in the Mv1Lu positive control, the MCF-10F nontumorigenic, and the tumorigenic MDA-MB231 cell lines. The other cell lines have little or no T β R-II protein present in the membrane fraction.

    Journal: Gene Expression

    Article Title: Responsiveness to Transforming Growth Factor-β (TGF-β)-Mediated Growth Inhibition Is a Function of Membrane-Bound TGF-β Type II Receptor in Human Breast Cancer Cells

    doi:

    Figure Lengend Snippet: T β R-II protein expression in total lysate, cytosol, and membrane fractions from human breast cell lines. Aliquots (10 μg) of total cell lysate and fractionated protein were analyzed by SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% nonfat dry milk in PBS-T, and incubated with T β R-II antibody. Bound antibodies were detected by incubation with peroxidase-conjugated antirabbit immunoglobulin and enhanced chemiluminscent autoradiography. A 70-kDa band corresponding to the size expected for T β R-II protein was observed in all cell lines in total lysate and the cytosolic fraction. The membrane fractions demonstrate the clear presence of T β R-II protein in the Mv1Lu positive control, the MCF-10F nontumorigenic, and the tumorigenic MDA-MB231 cell lines. The other cell lines have little or no T β R-II protein present in the membrane fraction.

    Article Snippet: Aliquots of protein (10 μg) were heated to 100°C in Tris-Glycine SDS sample buffer (63 mM Tris HCl, pH 7.4, 10% glycerol, 2% SDS, 0.0025% bromphenol blue) (Novex) and 5% β-mercaptoethanol (Sigma) and separated by SDS-PAGE electrophoresis on precast 8% Tris-Glycine gels (Novex) and transferred to Immobilon P membranes (Millipore, Bedford, MA).

    Techniques: Expressing, SDS Page, Electrophoresis, Incubation, Autoradiography, Positive Control, Multiple Displacement Amplification

    T β R-I protein expression in total lysate, cytosol, and membrane fractions from human breast cell lines. Aliquots (10 μg) of total cell lysate and fractionated protein were analyzed by SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% nonfat dry milk in PBS-T, and incubated with T β R-I antibody. Bound antibodies were detected by incubation with peroxidase-conjugated antirabbit immunoglobulin and enhanced chemiluminscent autoradiography. A 55-kDa band corresponding to the presence of T β R-I protein in total lysate, cytosolic, and membrane-bound protein fractions was observed. Membrane-bound fractions (and to a lesser extent the total cellular lysate) contain two (or three in the case of MCF-7) bands of close molecular weight, the significance of which remains to be determined.

    Journal: Gene Expression

    Article Title: Responsiveness to Transforming Growth Factor-β (TGF-β)-Mediated Growth Inhibition Is a Function of Membrane-Bound TGF-β Type II Receptor in Human Breast Cancer Cells

    doi:

    Figure Lengend Snippet: T β R-I protein expression in total lysate, cytosol, and membrane fractions from human breast cell lines. Aliquots (10 μg) of total cell lysate and fractionated protein were analyzed by SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% nonfat dry milk in PBS-T, and incubated with T β R-I antibody. Bound antibodies were detected by incubation with peroxidase-conjugated antirabbit immunoglobulin and enhanced chemiluminscent autoradiography. A 55-kDa band corresponding to the presence of T β R-I protein in total lysate, cytosolic, and membrane-bound protein fractions was observed. Membrane-bound fractions (and to a lesser extent the total cellular lysate) contain two (or three in the case of MCF-7) bands of close molecular weight, the significance of which remains to be determined.

    Article Snippet: Aliquots of protein (10 μg) were heated to 100°C in Tris-Glycine SDS sample buffer (63 mM Tris HCl, pH 7.4, 10% glycerol, 2% SDS, 0.0025% bromphenol blue) (Novex) and 5% β-mercaptoethanol (Sigma) and separated by SDS-PAGE electrophoresis on precast 8% Tris-Glycine gels (Novex) and transferred to Immobilon P membranes (Millipore, Bedford, MA).

    Techniques: Expressing, SDS Page, Electrophoresis, Incubation, Autoradiography, Molecular Weight

    Interactions between filamentous actin, GFP-Lpd (850–1250aa), and GFP-LZ-Lpd (850–1250aa) measured by cosedimentation at different buffer ionic strengths. ( A ) Monomeric GFP-Lpd 850−1250aa and dimeric GFP-LZ-Lpd 850−1250aa interact with filamentous actin in the presence of 50, 100, 150 mM KCl. SDS-PAGE from three experiments showing the cosedimentation of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa (monomeric protein concentration). Buffer composition is 20 mM HEPES [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl 2 , 0.5 mM EGTA. ( B ) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). ( C ) SDS-PAGE as in Figure 1H , showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). ( D ) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl 2 , and 0.5 mM EGTA. ( C , D ) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification. DOI: http://dx.doi.org/10.7554/eLife.06585.004

    Journal: eLife

    Article Title: Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    doi: 10.7554/eLife.06585

    Figure Lengend Snippet: Interactions between filamentous actin, GFP-Lpd (850–1250aa), and GFP-LZ-Lpd (850–1250aa) measured by cosedimentation at different buffer ionic strengths. ( A ) Monomeric GFP-Lpd 850−1250aa and dimeric GFP-LZ-Lpd 850−1250aa interact with filamentous actin in the presence of 50, 100, 150 mM KCl. SDS-PAGE from three experiments showing the cosedimentation of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa (monomeric protein concentration). Buffer composition is 20 mM HEPES [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl 2 , 0.5 mM EGTA. ( B ) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). ( C ) SDS-PAGE as in Figure 1H , showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). ( D ) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl 2 , and 0.5 mM EGTA. ( C , D ) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification. DOI: http://dx.doi.org/10.7554/eLife.06585.004

    Article Snippet: Actin was then combined with 2× TIRF imaging buffer (20 µl volume) and 4× protein (i.e., VASP, profilin, Lpd) (10 µl volume) resulting in a final buffer composition of 20 mM HEPES [pH 7], 50–100 mM KCl, 1 mM MgCl2 , 1 mM EGTA, 0.2% methylcellulose cP400, 1 mg/ml BSA, 1 mM ATP, 20 mM BME, 1 mM Trolox (Sigma, Cat# 238813), 20 mM glucose, 125 µg/ml glucose oxidase (Serva, #22780.01 Aspergillus niger ), and 20 µg/ml catalase (Sigma, #C40-100MG Bovine Liver).

    Techniques: SDS Page, Protein Concentration, Sedimentation, Concentration Assay, Incubation, Purification

    ITC results for binding of (A) WT RelA-NLS(293-321) (B) F309A RelA-NLS(293-321). (C) WT RelA(190-321)/p50(248-350) (D) F309A RelA(190-321)/p50(248-350) to IκBα(67-206) in 50 mM NaCl, 25 mM Tris, pH 7.5, 0.5mM EDTA, and 0.5mM sodium azide

    Journal: Journal of molecular biology

    Article Title: The RelA nuclear localization signal folds upon binding to I?B?

    doi: 10.1016/j.jmb.2010.10.055

    Figure Lengend Snippet: ITC results for binding of (A) WT RelA-NLS(293-321) (B) F309A RelA-NLS(293-321). (C) WT RelA(190-321)/p50(248-350) (D) F309A RelA(190-321)/p50(248-350) to IκBα(67-206) in 50 mM NaCl, 25 mM Tris, pH 7.5, 0.5mM EDTA, and 0.5mM sodium azide

    Article Snippet: Two one-liter growths were inoculated with the M9(90% D2 O) culture and induced at OD600 = 0.6 with 0.2 mM IPTG for 24 hours at 18° C. For all IκBα(67-206) preparations, the cells were collected by centrifugation at 5000 rpm for 30 minutes and resuspended in 70 mL/liter of culture of 25 mM Tris (pH7.5), 50 mM NaCl, 0.5 mM EDTA, 10 mM β-mercaptoethanol, 0.3 mM PMSF, and protease inhibitor cocktail (Sigma) and lysed by sonication on ice.

    Techniques: Binding Assay

    Presence of p14 in plasma membrane and soluble part of acrosome. ( A ) Western blot analysis showing the expression of p14 in membrane and cytosolic fractions of sperm and whole cell lysate. ( B ) Expression of the p14 in the cytosolic fraction of epididymis and cauda epididymal plasma. ( C ) Western blot showing the presence of p14 in plasma membranes and soluble components of the acrosome, acrosomal matrix and sperm cell lysate. ( D ) Immunoblot with a peripheral plasma membrane protein extract prepared from washed caudal epididymis following treatment with AES solution and cell lysate. Proteins were extracted from caudal sperm with 0.625% Triton-X-100 solution for immunoblotting (details in methods ). Experiments were repeated four times.

    Journal: PLoS ONE

    Article Title: Elucidation of the Involvement of p14, a Sperm Protein during Maturation, Capacitation and Acrosome Reaction of Caprine Spermatozoa

    doi: 10.1371/journal.pone.0030552

    Figure Lengend Snippet: Presence of p14 in plasma membrane and soluble part of acrosome. ( A ) Western blot analysis showing the expression of p14 in membrane and cytosolic fractions of sperm and whole cell lysate. ( B ) Expression of the p14 in the cytosolic fraction of epididymis and cauda epididymal plasma. ( C ) Western blot showing the presence of p14 in plasma membranes and soluble components of the acrosome, acrosomal matrix and sperm cell lysate. ( D ) Immunoblot with a peripheral plasma membrane protein extract prepared from washed caudal epididymis following treatment with AES solution and cell lysate. Proteins were extracted from caudal sperm with 0.625% Triton-X-100 solution for immunoblotting (details in methods ). Experiments were repeated four times.

    Article Snippet: Materials and Methods Sodium lactate, bovine serum albumins, calcium ionophore A23187, Triton-X-100, EDTA, EGTA, beta-mercaptoethanol, SDS, protease inhibitor cocktail, Na3 VO4 , glycerol, leupeptin, aprotinin, PMSF, PVDF membrane, goat-antirabbit IgG (alkaline phosphatase conjugated), and FITC-conjugated goat anti-rabbit-IgG, Rose-Bengal stain, kanamycin, penicillamine, TPCK, TLCK, DEAE-cellulose, thioglycerol, TEMED, cacodylate were obtained from Sigma Chemicals, USA.

    Techniques: Western Blot, Expressing

    STAP-2 enhances proliferation of DU145 cells in vitro and in vivo . A , qPCR analysis for mRNA levels of STAP-2 in various cancer cell lines and human tissues. B and C , expression levels of STAP-2 in STAP-2–knockdown DU145 cells were determined by qPCR ( B ) and immunoblotting ( IB ) ( C ). TCL , total cell lysates. D , viable cells of control or STAP-2 shRNA–expressing DU145 cells were measured by a WST assay. E , control or STAP-2 shRNA–expressing DU145 cells were cultured in DMEM containing 1% FBS for the indicated times, and then the surviving cells were measured by a WST assay. F , STAP-2–knockdown DU145 cells were transfected with pcDNA3.1-Myc-STAP-2, and then their cell proliferation was measured by a WST assay at 72 h post-transfection, as in D . An aliquot of total cell lysates was immunoblotted by anti-Myc and anti-actin antibodies. G , expression levels of STAP-2 in STAP-2–knockdown LNCaP cells were determined by immunoblotting. H , viable cells of control or STAP-2 shRNA–expressing LNCaP cells were measured by a WST assay. I , DU145 cells (5.0 × 10 6 cells) were subcutaneously injected into BALB/c nude mice (day 0), and tumor volume was measured. Tumors were established in 5 of 5 mice (shControl) and 3 of 5 mice (shSTAP-2). J , weight of tumors and spleens from the tumor-bearing mice at day 33. n = 3; mean values and S.E. ( error bars ) are depicted. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: STAP-2 protein promotes prostate cancer growth by enhancing epidermal growth factor receptor stabilization

    doi: 10.1074/jbc.M117.802884

    Figure Lengend Snippet: STAP-2 enhances proliferation of DU145 cells in vitro and in vivo . A , qPCR analysis for mRNA levels of STAP-2 in various cancer cell lines and human tissues. B and C , expression levels of STAP-2 in STAP-2–knockdown DU145 cells were determined by qPCR ( B ) and immunoblotting ( IB ) ( C ). TCL , total cell lysates. D , viable cells of control or STAP-2 shRNA–expressing DU145 cells were measured by a WST assay. E , control or STAP-2 shRNA–expressing DU145 cells were cultured in DMEM containing 1% FBS for the indicated times, and then the surviving cells were measured by a WST assay. F , STAP-2–knockdown DU145 cells were transfected with pcDNA3.1-Myc-STAP-2, and then their cell proliferation was measured by a WST assay at 72 h post-transfection, as in D . An aliquot of total cell lysates was immunoblotted by anti-Myc and anti-actin antibodies. G , expression levels of STAP-2 in STAP-2–knockdown LNCaP cells were determined by immunoblotting. H , viable cells of control or STAP-2 shRNA–expressing LNCaP cells were measured by a WST assay. I , DU145 cells (5.0 × 10 6 cells) were subcutaneously injected into BALB/c nude mice (day 0), and tumor volume was measured. Tumors were established in 5 of 5 mice (shControl) and 3 of 5 mice (shSTAP-2). J , weight of tumors and spleens from the tumor-bearing mice at day 33. n = 3; mean values and S.E. ( error bars ) are depicted. *, p

    Article Snippet: DU145 and HEK293T cells were cultured in DMEM (Sigma) supplemented with 10% FBS (Sigma) and 0.05 m m 2-mercaptoethanol (Nacalai Tesque) at 37 °C in a humidified 5% CO2 , 95% air atmosphere.

    Techniques: In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Expressing, shRNA, WST Assay, Cell Culture, Transfection, Injection, Mouse Assay

    Analysis of 5-mC and 5-hmC during embryonic stem cell differentiation to embryoid body. A , LIF withdrawal leading to embryonic stem cell marker Oct4 and Nanog repression is shown. Shown is a Western blot of ES cell extract after LIF withdrawal along with NIH3T3 (3T3), as shown on the top , with the indicated antibodies on the left . Gapdh is the loading control. B , Oct4 and Nanog gene are methylated upon embryoid body formation. Day post-LIF withdrawal is on the left along with NIH3T3. UDP-Glc and β-GT are shown on the top along with restriction enzyme MspI ( M ) and HpaII ( H ) cleavage along with control ( C ). C , 5-hmC detection during embryoid body formation is shown. Day post-LIF withdrawal is shown on the left. D , global methylation analysis using LC-MS during embryoid body formation is shown. Each analysis point was performed in duplicate. Deoxycytosine (C), 5-methyldeoxycytosine (5-mC or mC ) and 5-hydroxymethyldeoxycytosine (5-hmC or hmC ) are shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes *

    doi: 10.1074/jbc.M110.217083

    Figure Lengend Snippet: Analysis of 5-mC and 5-hmC during embryonic stem cell differentiation to embryoid body. A , LIF withdrawal leading to embryonic stem cell marker Oct4 and Nanog repression is shown. Shown is a Western blot of ES cell extract after LIF withdrawal along with NIH3T3 (3T3), as shown on the top , with the indicated antibodies on the left . Gapdh is the loading control. B , Oct4 and Nanog gene are methylated upon embryoid body formation. Day post-LIF withdrawal is on the left along with NIH3T3. UDP-Glc and β-GT are shown on the top along with restriction enzyme MspI ( M ) and HpaII ( H ) cleavage along with control ( C ). C , 5-hmC detection during embryoid body formation is shown. Day post-LIF withdrawal is shown on the left. D , global methylation analysis using LC-MS during embryoid body formation is shown. Each analysis point was performed in duplicate. Deoxycytosine (C), 5-methyldeoxycytosine (5-mC or mC ) and 5-hydroxymethyldeoxycytosine (5-hmC or hmC ) are shown.

    Article Snippet: Cell Culture and E14 Embryonic Stem (ES) Cell Differentiation to Embryoid Bodies ES cells were cultured as described with GMEM (Invitrogen) media containing 10% FBS (Gemcell), 1% non-essential amino acids (NEAA) (Hyclone), 1% sodium pyruvate (Invitrogen), 50 μm β-mercaptoethanol (Sigma), and 1× LIF (Millipore).

    Techniques: Cell Differentiation, Marker, Western Blot, Methylation, Gas Chromatography, Liquid Chromatography with Mass Spectroscopy

    sPLA 2 s induce the release of VEGF-A and VEGF-C from HLMs. A and B , HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (Control) or with the indicated concentrations of hGIIA, hGX, or LPS. VEGF-A ( A ) and VEGF-C ( B ) release was determined by ELISA. Data are mean ± SEM of four experiments. * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Production of Vascular Endothelial Growth Factors from Human Lung Macrophages Induced by Group IIA and Group X Secreted Phospholipases A2

    doi: 10.4049/jimmunol.0902501

    Figure Lengend Snippet: sPLA 2 s induce the release of VEGF-A and VEGF-C from HLMs. A and B , HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (Control) or with the indicated concentrations of hGIIA, hGX, or LPS. VEGF-A ( A ) and VEGF-C ( B ) release was determined by ELISA. Data are mean ± SEM of four experiments. * p

    Article Snippet: In selected experiments, HLMs (6 × 106 /4 ml) were incubated (37°C, 24 h) with RPMI 1640 alone or hGX (3 μg/ml), and at the end of incubation, cell-free supernatants were concentrated by centrifugation (4000 × g , 4°C, 15 min) over Amicon Ultra-4 filter units (Millipore, Billerica, CA) with a cutoff of 10 kDa.

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay

    Supernatants of HLMs activated by sPLA 2 induce an angiogenic response in the chick embryo CAM. Chick embryo CAMs at day 8 of incubation were implanted with gelatin sponges adsorbed with RPMI 1640 alone ( A ), supernatants of HLMs kept in culture for 24 h at 37°C with RPMI 1640 alone (control, B ), with Me-Indoxam alone (10 μM, C ), with hGX alone (3 μg/ml, D ), or with the combination hGX plus Me-Indoxam ( E ). At day 12 of incubation, CAMs were examined as described under Materials and Methods . A–E , original magnification ×50. The experiment shown is representative of three separate experiments. In F , data are expressed as number of vessels at the sponge-CAM boundary and are the mean ± SEM of three experiments. * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Production of Vascular Endothelial Growth Factors from Human Lung Macrophages Induced by Group IIA and Group X Secreted Phospholipases A2

    doi: 10.4049/jimmunol.0902501

    Figure Lengend Snippet: Supernatants of HLMs activated by sPLA 2 induce an angiogenic response in the chick embryo CAM. Chick embryo CAMs at day 8 of incubation were implanted with gelatin sponges adsorbed with RPMI 1640 alone ( A ), supernatants of HLMs kept in culture for 24 h at 37°C with RPMI 1640 alone (control, B ), with Me-Indoxam alone (10 μM, C ), with hGX alone (3 μg/ml, D ), or with the combination hGX plus Me-Indoxam ( E ). At day 12 of incubation, CAMs were examined as described under Materials and Methods . A–E , original magnification ×50. The experiment shown is representative of three separate experiments. In F , data are expressed as number of vessels at the sponge-CAM boundary and are the mean ± SEM of three experiments. * p

    Article Snippet: In selected experiments, HLMs (6 × 106 /4 ml) were incubated (37°C, 24 h) with RPMI 1640 alone or hGX (3 μg/ml), and at the end of incubation, cell-free supernatants were concentrated by centrifugation (4000 × g , 4°C, 15 min) over Amicon Ultra-4 filter units (Millipore, Billerica, CA) with a cutoff of 10 kDa.

    Techniques: Chick Chorioallantoic Membrane Assay, Incubation

    sPLA 2 -induced production of VEGF-A is independent from enzymatic activity and involves a receptor-mediated activation of HLMs. A , Effect of H48Q mutants of sPLA 2 s on VEGF-A release. HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (ctr), with the wt hGIIA and hGX, or the mutants H48Q of hGIIA and hGX. VEGF-A release was determined by ELISA. Data are mean ± SEM of three experiments. * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Production of Vascular Endothelial Growth Factors from Human Lung Macrophages Induced by Group IIA and Group X Secreted Phospholipases A2

    doi: 10.4049/jimmunol.0902501

    Figure Lengend Snippet: sPLA 2 -induced production of VEGF-A is independent from enzymatic activity and involves a receptor-mediated activation of HLMs. A , Effect of H48Q mutants of sPLA 2 s on VEGF-A release. HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (ctr), with the wt hGIIA and hGX, or the mutants H48Q of hGIIA and hGX. VEGF-A release was determined by ELISA. Data are mean ± SEM of three experiments. * p

    Article Snippet: In selected experiments, HLMs (6 × 106 /4 ml) were incubated (37°C, 24 h) with RPMI 1640 alone or hGX (3 μg/ml), and at the end of incubation, cell-free supernatants were concentrated by centrifugation (4000 × g , 4°C, 15 min) over Amicon Ultra-4 filter units (Millipore, Billerica, CA) with a cutoff of 10 kDa.

    Techniques: Activity Assay, Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Adenosine induces an angiogenic switch in sPLA 2 -activated HLMs. A , Effect of NECA on hGX-induced release of VEGF-A. HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (ctr), NECA alone (0.1–10 μM), hGX alone (3 μg/ml), or the indicated combinations of hGX plus NECA. VEGF-A releasewas determined by ELISA. Data are the mean ± SEM of three experiments. * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Production of Vascular Endothelial Growth Factors from Human Lung Macrophages Induced by Group IIA and Group X Secreted Phospholipases A2

    doi: 10.4049/jimmunol.0902501

    Figure Lengend Snippet: Adenosine induces an angiogenic switch in sPLA 2 -activated HLMs. A , Effect of NECA on hGX-induced release of VEGF-A. HLMs were incubated (37°C, 24 h) with RPMI 1640 alone (ctr), NECA alone (0.1–10 μM), hGX alone (3 μg/ml), or the indicated combinations of hGX plus NECA. VEGF-A releasewas determined by ELISA. Data are the mean ± SEM of three experiments. * p

    Article Snippet: In selected experiments, HLMs (6 × 106 /4 ml) were incubated (37°C, 24 h) with RPMI 1640 alone or hGX (3 μg/ml), and at the end of incubation, cell-free supernatants were concentrated by centrifugation (4000 × g , 4°C, 15 min) over Amicon Ultra-4 filter units (Millipore, Billerica, CA) with a cutoff of 10 kDa.

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay

    In vitro copper reductase assay with the Xenopus laevis H3-H4 tetramer. (A) Standard curve for Cu 1+ detection for copper reductase assay conditions used with X. laevis tetramers with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:10 in presence of 2 mM β-Mercaptoethanol as reducing agent (see Methods). Error bars indicate mean ± SD of three replicate measurements. (B) Same as (A) but for reactions with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:15 (see Methods). (C) Progress curve of copper reduction using 30 µM TCEP as the electron donor with 1 µM of X. laevis tetramer, equimolar monomeric X. laevis histone H3, and equimolar monomeric S. cerevisiae histone H2A. (D) Henri-Michaelis-Menten curves of initial velocities of reactions with the X. laevis tetramer. (E) Calculated enzymatic parameters of the tetramer from the curve in (D). (F) Same as (C) but with X. laevis tetramer, or equimolar RNase A or DTT. (G) Same as (A) but for reactions with increasing amounts of CuCl 2 -ADA pH 8 at a ratio 1:1 (see Methods). (H) Same as (A) but for reactions with increasing amounts of CuCl 2 -NTA pH 7.6 at a ratio 1:1 (see Methods). (I) Progress curves of copper reduction by 5 µM of the indicated X. laevis tetramers in presence of 200 µM TCEP and 0.5 mM CuCl 2 in 0.5 mM NTA pH 7.6.

    Journal: bioRxiv

    Article Title: The Histone H3-H4 Tetramer is a Copper Reductase Enzyme

    doi: 10.1101/350652

    Figure Lengend Snippet: In vitro copper reductase assay with the Xenopus laevis H3-H4 tetramer. (A) Standard curve for Cu 1+ detection for copper reductase assay conditions used with X. laevis tetramers with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:10 in presence of 2 mM β-Mercaptoethanol as reducing agent (see Methods). Error bars indicate mean ± SD of three replicate measurements. (B) Same as (A) but for reactions with increasing amounts of CuCl 2 -Tricine pH 7.5 at a ratio 1:15 (see Methods). (C) Progress curve of copper reduction using 30 µM TCEP as the electron donor with 1 µM of X. laevis tetramer, equimolar monomeric X. laevis histone H3, and equimolar monomeric S. cerevisiae histone H2A. (D) Henri-Michaelis-Menten curves of initial velocities of reactions with the X. laevis tetramer. (E) Calculated enzymatic parameters of the tetramer from the curve in (D). (F) Same as (C) but with X. laevis tetramer, or equimolar RNase A or DTT. (G) Same as (A) but for reactions with increasing amounts of CuCl 2 -ADA pH 8 at a ratio 1:1 (see Methods). (H) Same as (A) but for reactions with increasing amounts of CuCl 2 -NTA pH 7.6 at a ratio 1:1 (see Methods). (I) Progress curves of copper reduction by 5 µM of the indicated X. laevis tetramers in presence of 200 µM TCEP and 0.5 mM CuCl 2 in 0.5 mM NTA pH 7.6.

    Article Snippet: Standard curves for each condition were produced using 2 mM β-Mercaptoethanol to ensure complete reduction of Cu2+ .

    Techniques: In Vitro, Reductase Assay

    PPM1H dephosphorylates Rab10 in vitro. ( A ) The indicated amounts of recombinant wild-type and mutant PPM1H (with a His-Sumo N-terminal tag, expressed in E. coli ) were incubated in vitro with 2.5 µg pT73 ~60% phosphorylated Rab10[1–181, GDP bound] for 30 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.0 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by phos-tag gel electrophoresis that separates phosphorylated and dephosphorylated Rab10. Gel was stained with Instant Blue Coomassie. Bands corresponding to phosphorylated and non-phosphorylated Rab10 are marked with open (○) and closed (●) circles respectively. ( B ) As in ( A ) except that a time-course assay was performed using 2.5 µg pT73 phosphorylated Rab10[1–181, GDP bound] and 40 ng wild-type or mutant PPM1H for the indicated times. ( C ) As in ( A ) except that PPM1J was assessed. ( D ) As in ( A ) except and PPM1M was assessed.

    Journal: eLife

    Article Title: PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

    doi: 10.7554/eLife.50416

    Figure Lengend Snippet: PPM1H dephosphorylates Rab10 in vitro. ( A ) The indicated amounts of recombinant wild-type and mutant PPM1H (with a His-Sumo N-terminal tag, expressed in E. coli ) were incubated in vitro with 2.5 µg pT73 ~60% phosphorylated Rab10[1–181, GDP bound] for 30 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.0 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by phos-tag gel electrophoresis that separates phosphorylated and dephosphorylated Rab10. Gel was stained with Instant Blue Coomassie. Bands corresponding to phosphorylated and non-phosphorylated Rab10 are marked with open (○) and closed (●) circles respectively. ( B ) As in ( A ) except that a time-course assay was performed using 2.5 µg pT73 phosphorylated Rab10[1–181, GDP bound] and 40 ng wild-type or mutant PPM1H for the indicated times. ( C ) As in ( A ) except that PPM1J was assessed. ( D ) As in ( A ) except and PPM1M was assessed.

    Article Snippet: The protein was eluted with 4 × 1 resin volume of elution buffer (30 mM Tris pH 7.5, 3% (by vol) glycerol, 0.4 M imidazole, 8 mM 2-mercaptoethanol, 1.2 mM MgCl2 , 0.02% (by vol) Brij35, 0.6 µM GTP-gamma-S) and dialyzed against 50 mM Tris pH 7.5, 10% (by vol) glycerol, 250 mM NaCl, 14 mM 2-mercaptoethanol, 2 mM MgCl2 , 0.6 µM GTP-gamma-S in the presence of 60 Units of Thrombin (Sigma-Aldrich T1063-1KU).

    Techniques: In Vitro, Recombinant, Mutagenesis, Incubation, Nucleic Acid Electrophoresis, Staining

    LPS of B . pseudomallei signals solely via murine TLR4. Murine whole blood (A) and peritoneal macrophages (B) harvested from wild-type (WT), TLR2 -/- and TLR4 -/- mice were stimulated for 24h with RPMI 1640 + 10% FCS medium, LPS of E . coli 0111:B4 (100 ng/ml), LPS of B . pseudomallei (100 ng/ml), heat-killed B . pseudomallei 1026b (100 ng/ml), or its O-antigen lacking mutant SRM117 (both 10 7 CFU/ml) before measurement of murine TNF-α (n = 4). Following a Kruskal-Wallis test, Mann-Whitney U-tests were performed. Data are means ± SEM. Results are representative of two or three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Differential Toll-Like Receptor-Signalling of Burkholderia pseudomallei Lipopolysaccharide in Murine and Human Models

    doi: 10.1371/journal.pone.0145397

    Figure Lengend Snippet: LPS of B . pseudomallei signals solely via murine TLR4. Murine whole blood (A) and peritoneal macrophages (B) harvested from wild-type (WT), TLR2 -/- and TLR4 -/- mice were stimulated for 24h with RPMI 1640 + 10% FCS medium, LPS of E . coli 0111:B4 (100 ng/ml), LPS of B . pseudomallei (100 ng/ml), heat-killed B . pseudomallei 1026b (100 ng/ml), or its O-antigen lacking mutant SRM117 (both 10 7 CFU/ml) before measurement of murine TNF-α (n = 4). Following a Kruskal-Wallis test, Mann-Whitney U-tests were performed. Data are means ± SEM. Results are representative of two or three independent experiments. * P

    Article Snippet: The murine alveolar macrophage cell line MH-S was grown in RPMI 1640 medium supplemented with 10% FCS, 1% L-glutamine, 1% PenStrep and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich) and seeded at 1.5 x106 /ml, followed by stimulation with the stimuli as described above in the presence or absence of LPS-binding polymyxin B (25–100 ug/ml; Sigma-Aldrich).

    Techniques: Mouse Assay, Mutagenesis, MANN-WHITNEY

    Successful extraction and purification of B . pseudomallei- LPS. LPS from B . pseudomallei 1026b was purified by a modified hot phenol-water extraction method including proteinase K treatment. 10 μl of LPS (0.5 mg/ml) was fractionated by SDS-PAGE electrophoresis followed by silver staining (A) which showed the characteristic ladder pattern of LPS banding of Gram-negative bacteria, without any indications of protein contamination (detection limit of 0.5–5 ng) and Coomassie blue staining (B) which demonstrated no protein contamination as well (detection limit 50 ng). Contamination of the extracted LPS was also assessed by the addition of LPS-binding Polymyxin B (PMB) (C). For this purpose, the murine alveolar macrophage cell line MH-S was stimulated with RPMI 1640 medium [ 56 ], LPS of E . coli 0111:B4 or B . pseudomallei 1026b and incubated with increasing concentrations of PMB for 6h, followed by TNF-α measurement in the supernatant. Results are representative for three independent experiments. (M = ladder, B.ps = B . pseudomallei -LPS)

    Journal: PLoS ONE

    Article Title: Differential Toll-Like Receptor-Signalling of Burkholderia pseudomallei Lipopolysaccharide in Murine and Human Models

    doi: 10.1371/journal.pone.0145397

    Figure Lengend Snippet: Successful extraction and purification of B . pseudomallei- LPS. LPS from B . pseudomallei 1026b was purified by a modified hot phenol-water extraction method including proteinase K treatment. 10 μl of LPS (0.5 mg/ml) was fractionated by SDS-PAGE electrophoresis followed by silver staining (A) which showed the characteristic ladder pattern of LPS banding of Gram-negative bacteria, without any indications of protein contamination (detection limit of 0.5–5 ng) and Coomassie blue staining (B) which demonstrated no protein contamination as well (detection limit 50 ng). Contamination of the extracted LPS was also assessed by the addition of LPS-binding Polymyxin B (PMB) (C). For this purpose, the murine alveolar macrophage cell line MH-S was stimulated with RPMI 1640 medium [ 56 ], LPS of E . coli 0111:B4 or B . pseudomallei 1026b and incubated with increasing concentrations of PMB for 6h, followed by TNF-α measurement in the supernatant. Results are representative for three independent experiments. (M = ladder, B.ps = B . pseudomallei -LPS)

    Article Snippet: The murine alveolar macrophage cell line MH-S was grown in RPMI 1640 medium supplemented with 10% FCS, 1% L-glutamine, 1% PenStrep and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich) and seeded at 1.5 x106 /ml, followed by stimulation with the stimuli as described above in the presence or absence of LPS-binding polymyxin B (25–100 ug/ml; Sigma-Aldrich).

    Techniques: Purification, Modification, SDS Page, Electrophoresis, Silver Staining, Staining, Binding Assay, Incubation

    LPS of B . pseudomallei signals via both TLR2 and TLR4 in in vitro human models. Human Embryonic Kidney (HEK)-293 cells, stably transfected with either CD14-TLR2 or CD14-TLR4/MD2 were stimulated with purified LPS of B . pseudomallei 1026b (100 ng/ml), LPS of E . coli 0111:B4 (100 ng/ml), PAM3CSK4 (100 ng/ml) or DMEM+ 10% FCS for 6h (A) or 24h (B) before measurement of interleukin (IL)-8 in the supernatant (n = 3). Human whole blood was pre-treated for 30 minutes with respectively RPMI 1640 medium, anti-TLR2 antibody (2500 ng/ml), anti-TLR4 antibody (1000 ng/ml) or both antibodies and hereafter stimulated with purified LPS of B . pseudomallei 1026b (100 ng/ml), LPS of E . coli 0111:B4 (100 ng/ml), PAM3CSK4 (100 ng/ml) or RPMI 1640 for 6h after which TNF was measured (C) (n = 3). An additive effect of TLR2 on TLR4 mediated signalling induced by B . pseudomallei -LPS was observed. Data are presented as means ± SEM. Results of two or three independent experiments were pooled. Mann-Whitney- U tests were performed. * P

    Journal: PLoS ONE

    Article Title: Differential Toll-Like Receptor-Signalling of Burkholderia pseudomallei Lipopolysaccharide in Murine and Human Models

    doi: 10.1371/journal.pone.0145397

    Figure Lengend Snippet: LPS of B . pseudomallei signals via both TLR2 and TLR4 in in vitro human models. Human Embryonic Kidney (HEK)-293 cells, stably transfected with either CD14-TLR2 or CD14-TLR4/MD2 were stimulated with purified LPS of B . pseudomallei 1026b (100 ng/ml), LPS of E . coli 0111:B4 (100 ng/ml), PAM3CSK4 (100 ng/ml) or DMEM+ 10% FCS for 6h (A) or 24h (B) before measurement of interleukin (IL)-8 in the supernatant (n = 3). Human whole blood was pre-treated for 30 minutes with respectively RPMI 1640 medium, anti-TLR2 antibody (2500 ng/ml), anti-TLR4 antibody (1000 ng/ml) or both antibodies and hereafter stimulated with purified LPS of B . pseudomallei 1026b (100 ng/ml), LPS of E . coli 0111:B4 (100 ng/ml), PAM3CSK4 (100 ng/ml) or RPMI 1640 for 6h after which TNF was measured (C) (n = 3). An additive effect of TLR2 on TLR4 mediated signalling induced by B . pseudomallei -LPS was observed. Data are presented as means ± SEM. Results of two or three independent experiments were pooled. Mann-Whitney- U tests were performed. * P

    Article Snippet: The murine alveolar macrophage cell line MH-S was grown in RPMI 1640 medium supplemented with 10% FCS, 1% L-glutamine, 1% PenStrep and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich) and seeded at 1.5 x106 /ml, followed by stimulation with the stimuli as described above in the presence or absence of LPS-binding polymyxin B (25–100 ug/ml; Sigma-Aldrich).

    Techniques: In Vitro, Stable Transfection, Transfection, Purification, MANN-WHITNEY

    2D 1 H- 15 N TROSY spectrum of 0.2mM selectively 15 N-isoleucine-labeled arrestin-1 in 25 mM Bis-Tris, 150mM NaCl and 5mM mercaptoethanol, pH=6.5 at 308 K using a Bruker Avance 800MHz spectrometer. 11 out of 20 isoleucine residue peaks were assigned, as labeled

    Journal: Biochemistry

    Article Title: Elucidation of IP6 and Heparin Interaction Sites and Conformational Changes in Arrestin-1 by Solution NMR †

    doi: 10.1021/bi101596g

    Figure Lengend Snippet: 2D 1 H- 15 N TROSY spectrum of 0.2mM selectively 15 N-isoleucine-labeled arrestin-1 in 25 mM Bis-Tris, 150mM NaCl and 5mM mercaptoethanol, pH=6.5 at 308 K using a Bruker Avance 800MHz spectrometer. 11 out of 20 isoleucine residue peaks were assigned, as labeled

    Article Snippet: For each NMR experiment the buffer was exchanged from Tris (25 mM Tris-HCl, pH=7.5, 150 mM NaCl, 5 mM DTT, 1 mM EDTA) to Bis-Tris buffer (25 mM Bis-Tris, pH=6.5, 150 mM NaCl, 5 mM mercaptoethanol) using an ultrafiltration concentrator (Millipore, molecular weight cut-off=30 kDa) immediately before use.

    Techniques: Labeling

    2-D 1 H- 15 N TROSY spectrum of 0.2mM U- 2 H, 15 N-arrestin-1 in 25 mM Bis-Tris, 150 mM NaCl, 5 mM mercaptoethanol, pH=6.5 acquired at 308 K using a Bruker Avance 800MHz spectrometer. 152 assigned residues are labeled.

    Journal: Biochemistry

    Article Title: Elucidation of IP6 and Heparin Interaction Sites and Conformational Changes in Arrestin-1 by Solution NMR †

    doi: 10.1021/bi101596g

    Figure Lengend Snippet: 2-D 1 H- 15 N TROSY spectrum of 0.2mM U- 2 H, 15 N-arrestin-1 in 25 mM Bis-Tris, 150 mM NaCl, 5 mM mercaptoethanol, pH=6.5 acquired at 308 K using a Bruker Avance 800MHz spectrometer. 152 assigned residues are labeled.

    Article Snippet: For each NMR experiment the buffer was exchanged from Tris (25 mM Tris-HCl, pH=7.5, 150 mM NaCl, 5 mM DTT, 1 mM EDTA) to Bis-Tris buffer (25 mM Bis-Tris, pH=6.5, 150 mM NaCl, 5 mM mercaptoethanol) using an ultrafiltration concentrator (Millipore, molecular weight cut-off=30 kDa) immediately before use.

    Techniques: Labeling

    K45 of RIPK1 promotes necroptotic signaling. (a, b , e – h ) WT or RIPK1 K45A macrophages were treated as indicated in figure panels with LPS (100 ng/ml) and TNF α (50 ng/ml) with or without zVAD-fmk (50 μ M), and samples were evaluated by western blotting. Panels ( a ) and ( b ) shows western blotting at 3 h posttreatment. Panel ( h ) shows western blotting at 4 h posttreatment. ( c , d , i – k ) Densitometry of pooled western blottings from several experiments. ANOVA with Bonferroni post-test was used to measure statistical significance, * P

    Journal: Cell Death and Differentiation

    Article Title: K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo

    doi: 10.1038/cdd.2016.51

    Figure Lengend Snippet: K45 of RIPK1 promotes necroptotic signaling. (a, b , e – h ) WT or RIPK1 K45A macrophages were treated as indicated in figure panels with LPS (100 ng/ml) and TNF α (50 ng/ml) with or without zVAD-fmk (50 μ M), and samples were evaluated by western blotting. Panels ( a ) and ( b ) shows western blotting at 3 h posttreatment. Panel ( h ) shows western blotting at 4 h posttreatment. ( c , d , i – k ) Densitometry of pooled western blottings from several experiments. ANOVA with Bonferroni post-test was used to measure statistical significance, * P

    Article Snippet: Reagents used were: LPS (from E. coli 0111:B4, L3024, Sigma), TNF α (410-MT, R & D), IFN β (12400-1, PBL Interferon, Piscataway, NJ, USA), zVAD-fmk (627610, Millipore, Billerica, MA, USA), BP (S7015, Selleckchem, Cedarlane, Burlington, ON, Canada), SMAC 164 (gift from Shaomeng Wang), Nec-1 (9037, Sigma), and Nec1s (gift from Dr. Katey Rayner, University of Ottawa).

    Techniques: Western Blot

    Functionalization procedure of PLLA/PLLA-PEG-NH 2 nanofibers (brown). Step 1: Amino groups are activated by exposing nanofibers to saturated water vapor. Step 2: Carboxyl groups are activated by hydrolysis. Blue represents the loaded drug; red is rhodamine BSA; and green is FITC-BSA. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PEG, poly(ethylene glycol); PLLA, poly(L-lactide).

    Journal: International Journal of Nanomedicine

    Article Title: PLLA-PEG-TCH-labeled bioactive molecule nanofibers for tissue engineering

    doi: 10.2147/IJN.S23688

    Figure Lengend Snippet: Functionalization procedure of PLLA/PLLA-PEG-NH 2 nanofibers (brown). Step 1: Amino groups are activated by exposing nanofibers to saturated water vapor. Step 2: Carboxyl groups are activated by hydrolysis. Blue represents the loaded drug; red is rhodamine BSA; and green is FITC-BSA. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PEG, poly(ethylene glycol); PLLA, poly(L-lactide).

    Article Snippet: NaOH (≥98%), benzyltriethylammonium chloride (BTAC) (98%), BSA (≥96%), 2-mercaptoethanol, fluorescein isothiocyanate (FITC)-conjugated BSA, rhodamine isothiocyanate, and peptide RGDS were from Sigma-Aldrich (Oakville, ON).

    Techniques:

    Confocal images of PLLA/PLLA-PEG-NH 2 nanofibers functionalized with both FITC-BSA and rhodamine-BSA. ( A ) Image showing FITC-BSA, ( B ) image showing rhodamine-BSA, and ( C ) merged A and B. Bar: 20 μm. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PEG, poly(ethylene glycol); PLLA, poly(L-lactide).

    Journal: International Journal of Nanomedicine

    Article Title: PLLA-PEG-TCH-labeled bioactive molecule nanofibers for tissue engineering

    doi: 10.2147/IJN.S23688

    Figure Lengend Snippet: Confocal images of PLLA/PLLA-PEG-NH 2 nanofibers functionalized with both FITC-BSA and rhodamine-BSA. ( A ) Image showing FITC-BSA, ( B ) image showing rhodamine-BSA, and ( C ) merged A and B. Bar: 20 μm. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PEG, poly(ethylene glycol); PLLA, poly(L-lactide).

    Article Snippet: NaOH (≥98%), benzyltriethylammonium chloride (BTAC) (98%), BSA (≥96%), 2-mercaptoethanol, fluorescein isothiocyanate (FITC)-conjugated BSA, rhodamine isothiocyanate, and peptide RGDS were from Sigma-Aldrich (Oakville, ON).

    Techniques: