Structured Review

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Focal accumulation of SIRT2 coincides with reduced MT acetylation in neurons. ( A ). SIRT2 antibodies label MTs in processes of cultured primary cortical neurons (DIV 10). Panels show SIRT2 (left), total α-tubulin (middle) and merged image (right) indicating overlapping staining of SIRT2 and α-tubulin. ( B and C ). Areas of high-level endogenous SIRT2 expression (arrows) overlap with reduced α-tubulin acetylation in perikarya (B) and processes (C) of cultured primary neurons (DIV 11). Panels show SIRT2 (left), acetylated α-tubulin (middle) and merged images. ( D ). Fractionation of proteins from cultured primary neurons shows that endogenous SIRT2.2 is found primarily in the insoluble pellet (P) fraction; in contrast, SIRT2.1 is found largely in the soluble (S) fraction. Samples from five independent cultures are shown. ( E ). Brain-enriched SIRT2 isoforms are associated with the insoluble fraction in transfected cells. Like SIRT2.2, a significant amount of SIRT2.3 is found in the <t>NP40-insoluble</t> (P) fraction of transiently transfected N2a cells. Endogenous SIRT2.1 in these cells is found almost exclusively in the soluble (S) fraction.
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Images

1) Product Images from "The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS"

Article Title: The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddr326

Focal accumulation of SIRT2 coincides with reduced MT acetylation in neurons. ( A ). SIRT2 antibodies label MTs in processes of cultured primary cortical neurons (DIV 10). Panels show SIRT2 (left), total α-tubulin (middle) and merged image (right) indicating overlapping staining of SIRT2 and α-tubulin. ( B and C ). Areas of high-level endogenous SIRT2 expression (arrows) overlap with reduced α-tubulin acetylation in perikarya (B) and processes (C) of cultured primary neurons (DIV 11). Panels show SIRT2 (left), acetylated α-tubulin (middle) and merged images. ( D ). Fractionation of proteins from cultured primary neurons shows that endogenous SIRT2.2 is found primarily in the insoluble pellet (P) fraction; in contrast, SIRT2.1 is found largely in the soluble (S) fraction. Samples from five independent cultures are shown. ( E ). Brain-enriched SIRT2 isoforms are associated with the insoluble fraction in transfected cells. Like SIRT2.2, a significant amount of SIRT2.3 is found in the NP40-insoluble (P) fraction of transiently transfected N2a cells. Endogenous SIRT2.1 in these cells is found almost exclusively in the soluble (S) fraction.
Figure Legend Snippet: Focal accumulation of SIRT2 coincides with reduced MT acetylation in neurons. ( A ). SIRT2 antibodies label MTs in processes of cultured primary cortical neurons (DIV 10). Panels show SIRT2 (left), total α-tubulin (middle) and merged image (right) indicating overlapping staining of SIRT2 and α-tubulin. ( B and C ). Areas of high-level endogenous SIRT2 expression (arrows) overlap with reduced α-tubulin acetylation in perikarya (B) and processes (C) of cultured primary neurons (DIV 11). Panels show SIRT2 (left), acetylated α-tubulin (middle) and merged images. ( D ). Fractionation of proteins from cultured primary neurons shows that endogenous SIRT2.2 is found primarily in the insoluble pellet (P) fraction; in contrast, SIRT2.1 is found largely in the soluble (S) fraction. Samples from five independent cultures are shown. ( E ). Brain-enriched SIRT2 isoforms are associated with the insoluble fraction in transfected cells. Like SIRT2.2, a significant amount of SIRT2.3 is found in the NP40-insoluble (P) fraction of transiently transfected N2a cells. Endogenous SIRT2.1 in these cells is found almost exclusively in the soluble (S) fraction.

Techniques Used: Cell Culture, Staining, Expressing, Fractionation, Transfection

2) Product Images from "C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins"

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052600

Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.
Figure Legend Snippet: Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.

Techniques Used: Binding Assay, Western Blot, Derivative Assay, Infection, Molecular Weight, Dot Blot, Mutagenesis, Construct, Lysis

Effect of E protein on the recognition of prM protein by human anti-prM mAbs. (A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5 . (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.
Figure Legend Snippet: Effect of E protein on the recognition of prM protein by human anti-prM mAbs. (A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5 . (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.

Techniques Used: Binding Assay, Dot Blot, Lysis, Derivative Assay, Transfection

3) Product Images from "Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1? and TRIF/IRF3"

Article Title: Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1? and TRIF/IRF3

Journal: PLoS ONE

doi: 10.1371/journal.pone.0098517

ZVAD reduced COX-2 protein expression in monocytes but not in macrophages. Macrophages and monocytes were obtained from Donor 1 and Donor 2 and were left untreated (lane 1) or were incubated with LPS at 20 ng/ml in the absence (lane 2) or in presence of ZVAD (lane 3) for 12 h. Cells were lysed in 1% NP40 and proteins were analyzed by SDS-PAGE (12 and 16 µg protein per lane for monocytes and macrophages, respectively) and Western Blotting with Abs against COX-2 (panels 1 and 3 from the top) or with β-actin as loading control (panels 2 and 4 from the top). Densitometry was performed on developed films and percent reduction of COX-2 protein expression in LPS-treated monocytes in the presence of ZVAD was calculated relative to the expression of COX-2 in monocytes treated with LPS alone.
Figure Legend Snippet: ZVAD reduced COX-2 protein expression in monocytes but not in macrophages. Macrophages and monocytes were obtained from Donor 1 and Donor 2 and were left untreated (lane 1) or were incubated with LPS at 20 ng/ml in the absence (lane 2) or in presence of ZVAD (lane 3) for 12 h. Cells were lysed in 1% NP40 and proteins were analyzed by SDS-PAGE (12 and 16 µg protein per lane for monocytes and macrophages, respectively) and Western Blotting with Abs against COX-2 (panels 1 and 3 from the top) or with β-actin as loading control (panels 2 and 4 from the top). Densitometry was performed on developed films and percent reduction of COX-2 protein expression in LPS-treated monocytes in the presence of ZVAD was calculated relative to the expression of COX-2 in monocytes treated with LPS alone.

Techniques Used: Expressing, Incubation, SDS Page, Western Blot

4) Product Images from "SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology"

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.033043

SIL1 is required to maintain ER proteostasis in quadriceps as mice age. (A) Wild-type and Sil1 Gt quadriceps lysates ( n =3-5 pooled), solubilized in 8 M urea, isolated from mice at the depicted ages were normalized for an equivalent amount of total protein, pooled and subjected to reducing SDS electrophoresis, followed by transfer to PVDF membranes, and blotting with the indicated immune reagents for ER chaperones and co-chaperones. GAPDH serves as a control for loading. (B) Wild-type and Sil1 Gt quadriceps obtained from mice at the designated ages were lysed with NP40 lysis buffer. The resulting NP40-soluble and NP40-insoluble fractions were processed for western blotting with BiP and calnexin antisera. HSC70 serves as a loading control for the NP40-soluble fraction. (C) Urea-solubilized quadriceps lysates derived from mice of the indicated genotypes and ages were probed with antibodies to proteins that traffic through the secretory pathway. (D) NP40-solubilized quadriceps lysates from 12-month-old mice were processed for western blotting to detect the insulin receptor precursor (pro-IR) and the mature subunit (IR-β). (E) Graphical representation of normalized mRNA fold changes of proteins probed in C from Sil1 Gt quadriceps, relative to wild-type quadriceps ( n =3) (set to 1; indicated by the dotted line) at 3 (gray) and 15 (blue) months. Error bars indicate means±s.e.m. Statistical differences were computed using unpaired, two-tailed Student's t -tests, and are indicated as * P ≤0.05, *** P ≤0.001 and **** P ≤0.0001.
Figure Legend Snippet: SIL1 is required to maintain ER proteostasis in quadriceps as mice age. (A) Wild-type and Sil1 Gt quadriceps lysates ( n =3-5 pooled), solubilized in 8 M urea, isolated from mice at the depicted ages were normalized for an equivalent amount of total protein, pooled and subjected to reducing SDS electrophoresis, followed by transfer to PVDF membranes, and blotting with the indicated immune reagents for ER chaperones and co-chaperones. GAPDH serves as a control for loading. (B) Wild-type and Sil1 Gt quadriceps obtained from mice at the designated ages were lysed with NP40 lysis buffer. The resulting NP40-soluble and NP40-insoluble fractions were processed for western blotting with BiP and calnexin antisera. HSC70 serves as a loading control for the NP40-soluble fraction. (C) Urea-solubilized quadriceps lysates derived from mice of the indicated genotypes and ages were probed with antibodies to proteins that traffic through the secretory pathway. (D) NP40-solubilized quadriceps lysates from 12-month-old mice were processed for western blotting to detect the insulin receptor precursor (pro-IR) and the mature subunit (IR-β). (E) Graphical representation of normalized mRNA fold changes of proteins probed in C from Sil1 Gt quadriceps, relative to wild-type quadriceps ( n =3) (set to 1; indicated by the dotted line) at 3 (gray) and 15 (blue) months. Error bars indicate means±s.e.m. Statistical differences were computed using unpaired, two-tailed Student's t -tests, and are indicated as * P ≤0.05, *** P ≤0.001 and **** P ≤0.0001.

Techniques Used: Mouse Assay, Isolation, Electrophoresis, Lysis, Western Blot, Derivative Assay, Two Tailed Test

5) Product Images from "BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum"

Article Title: BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028542

BAT3 associates with Derlin2. A. HA-Ri332 was synthesized in a rabbit reticulocyte lysate in the presence or absence of canine pancreatic microsomal membranes (MM). Following NP40-mediated lysis, Ri332 was retrieved by immunoprecipitation. The immunoprecipitate and input samples were blotted for either BAT3 or HA as indicated. B. 293T cells were subjected to NP40 lysis, followed by retrieval of the indicated proteins. Pre-immune serum served as a control. The eluates were blotted for BAT3, as were the input control samples. C. Immunofluorescence of Hela cells using antibodies against endogenous BAT3 (green) and Derlin2 (red). Scale bar = 5 µm. D. 293T cells were transiently transfected with the indicated constructs, subjected to NP40 lysis followed by an immunoprecipitation for Derlin2. Immunoprecipitates were blotted for BAT3. Input samples were blotted for BAT3 and Derlin2. The asterisk indicates non-specific polypeptides.
Figure Legend Snippet: BAT3 associates with Derlin2. A. HA-Ri332 was synthesized in a rabbit reticulocyte lysate in the presence or absence of canine pancreatic microsomal membranes (MM). Following NP40-mediated lysis, Ri332 was retrieved by immunoprecipitation. The immunoprecipitate and input samples were blotted for either BAT3 or HA as indicated. B. 293T cells were subjected to NP40 lysis, followed by retrieval of the indicated proteins. Pre-immune serum served as a control. The eluates were blotted for BAT3, as were the input control samples. C. Immunofluorescence of Hela cells using antibodies against endogenous BAT3 (green) and Derlin2 (red). Scale bar = 5 µm. D. 293T cells were transiently transfected with the indicated constructs, subjected to NP40 lysis followed by an immunoprecipitation for Derlin2. Immunoprecipitates were blotted for BAT3. Input samples were blotted for BAT3 and Derlin2. The asterisk indicates non-specific polypeptides.

Techniques Used: Synthesized, Lysis, Immunoprecipitation, Immunofluorescence, Transfection, Construct

TCRα is engaged by BAT3. 293T cells were transiently transfected with TCRα and either empty vector, UBX-EBV, or p97 QQ, and labeled overnight with [ 35 S] methionine/cysteine to achieve steady state labeling. Cells were harvested and subjected to NP40 lysis. The lysates were precleared using pre-immune serum and immobilized protein A. Lysates were adjusted for total levels of incorporated isotope and subjected to immunoprecipitation for TCRα. The captured protein was eluted in 1% SDS at 37°C followed by a second immunoprecipitation for the indicated proteins.
Figure Legend Snippet: TCRα is engaged by BAT3. 293T cells were transiently transfected with TCRα and either empty vector, UBX-EBV, or p97 QQ, and labeled overnight with [ 35 S] methionine/cysteine to achieve steady state labeling. Cells were harvested and subjected to NP40 lysis. The lysates were precleared using pre-immune serum and immobilized protein A. Lysates were adjusted for total levels of incorporated isotope and subjected to immunoprecipitation for TCRα. The captured protein was eluted in 1% SDS at 37°C followed by a second immunoprecipitation for the indicated proteins.

Techniques Used: Transfection, Plasmid Preparation, Labeling, Lysis, Immunoprecipitation

6) Product Images from "Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes"

Article Title: Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M067363

ANGPTL4-mediated loss of LPL protein occurs in a post-ER compartment. A: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 5 μg/ml brefeldin A for 2 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). B: Western blot of cell lysates of adipocytes that had been differentiated from stromal vascular fractions from WAT of Angptl4 −/− and wild-type mice. Cells were lysed in NP-40 lysis buffer. Western blots were probed with antibodies against LPL, HSP90 (as a loading control), and H2A (as a loading control). NP40S, NP40-soluble LPL; NP40P, NP40-precipitated LPL; s.e., short exposure; l.e., long exposure. C: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from the stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 10 μM monensin (Mon.) for 3 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). D: Western blot of EndoH-treated cell lysates from adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. The cells were treated with 20 μM E64D for 24 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). E: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 5 mM 3MA for 10 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). F: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions from WAT of Angptl4 −/− and wild-type mice. The cells had been treated with 10 mM leupeptin (Leup.) for 10 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). EndoH-resistant LPL (high-mannose oligosaccharides, ER LPL) is indicated with R; EndoH-sensitive LPL (complex oligosaccharides; Golgi and cell surface LPL) is indicated with S.
Figure Legend Snippet: ANGPTL4-mediated loss of LPL protein occurs in a post-ER compartment. A: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 5 μg/ml brefeldin A for 2 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). B: Western blot of cell lysates of adipocytes that had been differentiated from stromal vascular fractions from WAT of Angptl4 −/− and wild-type mice. Cells were lysed in NP-40 lysis buffer. Western blots were probed with antibodies against LPL, HSP90 (as a loading control), and H2A (as a loading control). NP40S, NP40-soluble LPL; NP40P, NP40-precipitated LPL; s.e., short exposure; l.e., long exposure. C: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from the stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 10 μM monensin (Mon.) for 3 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). D: Western blot of EndoH-treated cell lysates from adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. The cells were treated with 20 μM E64D for 24 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). E: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4 −/− and wild-type mice. Cells were treated with 5 mM 3MA for 10 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). F: Western blot of EndoH-treated cell lysates of adipocytes that had been differentiated from stromal vascular fractions from WAT of Angptl4 −/− and wild-type mice. The cells had been treated with 10 mM leupeptin (Leup.) for 10 h. Western blots were probed with antibodies against LPL and HSP90 (as a loading control). EndoH-resistant LPL (high-mannose oligosaccharides, ER LPL) is indicated with R; EndoH-sensitive LPL (complex oligosaccharides; Golgi and cell surface LPL) is indicated with S.

Techniques Used: Western Blot, Mouse Assay, Lysis

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Centrifugation:

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Blocking Assay:

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Incubation:

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Article Snippet: Western Blot Whole cell lysates from SW1116 were prepared in NP40 lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). .. After transfer, blocking of unspecific binding sites was achieved by incubation in TBST (50 mM Tris/HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.2) containing 5% skimmed milk.

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: .. The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche). .. In all cases, tissue lysates were electrophoresed under reducing conditions on gels ranging from 8% to 16%, or on 4% to 20% gradient gels (Mini-PROTEAN® TGX™ Precast Protein Gels, Bio-Rad, Inc., Hercules, CA), depending on the size of the examined protein.

Mass Spectrometry:

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: The pulverized tissue was lysed, as described for mass spectrometry analysis, normalized for equivalent protein concentration using a BCA Protein Assay Kit (Pierce–ThermoScientific, Rockford, IL) and samples from the indicated number of mice at the same age were pooled. .. The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche).

BIA-KA:

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: The pulverized tissue was lysed, as described for mass spectrometry analysis, normalized for equivalent protein concentration using a BCA Protein Assay Kit (Pierce–ThermoScientific, Rockford, IL) and samples from the indicated number of mice at the same age were pooled. .. The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche).

Western Blot:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: Paragraph title: Transfection, cell lysates, Western blot analysis and dot blot assay ... At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

Article Title: Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes
Article Snippet: Upon differentiation, adipocytes were lysed in NP40 lysis buffer [50 mM Tris-HCl (pH 8.0), 0.5% NP40, 150 mM NaCl, 5 mM MgCl2 ] supplemented with protease and phosphatase inhibitors (Roche). .. NP40S and NP40P fractions were then loaded onto SDS-PAGE for further analyses by Western blotting.

Article Title: Phosphorylation of MAVS/VISA by Nemo-like kinase (NLK) for degradation regulates the antiviral innate immune response
Article Snippet: Coimmunoprecipitation and immunoblot analyses HEK293T cells (1 × 106 ) were transfected or stimulated and harvested in 400 µL of NP40 lysis buffer (30 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1% NP40) with a protease inhibitor cocktail (Roche, Basel, Switzerland). .. The Clarity™ Western ECL Substrate System (Bio-Rad) was used for protein detection.

Article Title: TTC5 is required to prevent apoptosis of acute myeloid leukemia stem cells
Article Snippet: Paragraph title: Western blotting and immunoprecipitation ... For co-immunoprecipitation, cells were lysed in NP40 Lysis Buffer (50 mM Tris/HCl (pH 8.0), 50 mM KCl, 10 mM EDTA, 1% NP40, 6.25 mM NaF, 20 mM β -glycerophosphate, 1 mM DTT and 1 × Protease Inhibitor cocktail (Roche)).

Article Title: Synergistic Antitumor Effects of Endostar in Combination with Oxaliplatin via Inhibition of HIF and CXCR4 in the Colorectal Cell Line SW1116
Article Snippet: .. Western Blot Whole cell lysates from SW1116 were prepared in NP40 lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). ..

Article Title: miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs
Article Snippet: .. Western blot Cell lines and mouse pituitary tissues were lysed in NP40 lysis buffer: 250 mM NaCl, Tris 50 mM (pH 8.0), 5 mM EDTA, 0.5% NP-40 (v/v) and 2× Protease inhibitor tablets (Roche). .. Pituitary tissue samples were removed from −80°C storage immediately prior to use and homogenised in an appropriate volume of ice-cold NP40 lysis buffer.

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: Paragraph title: Western blotting ... The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche).

Transfection:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: Paragraph title: Transfection, cell lysates, Western blot analysis and dot blot assay ... At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

Article Title: Phosphorylation of MAVS/VISA by Nemo-like kinase (NLK) for degradation regulates the antiviral innate immune response
Article Snippet: .. Coimmunoprecipitation and immunoblot analyses HEK293T cells (1 × 106 ) were transfected or stimulated and harvested in 400 µL of NP40 lysis buffer (30 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1% NP40) with a protease inhibitor cocktail (Roche, Basel, Switzerland). .. Next, the supernatants were incubated with the indicated antibodies and Protein G beads (Roche) at 4 °C for 5 h. The beads were then washed three times with lysis buffer, and immunoprecipitants were eluted with SDS loading buffer and fractionated on SDS-PAGE gels.

Pulse Chase:

Article Title: BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum
Article Snippet: .. Immunoprecipitations, Pulse-Chase Experiments, and SDS-PAGE Cells were lysed in NP40 lysis buffer (0.5% NP40, 10 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2 , pH7.4) supplemented with a complete protease inhibitor cocktail (Roche) and 2.5 mM N-Ethylmaleimide. .. The immunoprecipiation was performed using 30 µl of immobilized rProtein A (IPA 300, Repligen) with the relevant antibodies, or with 12 µl anti-HA Affinity Matrix (3F10, Roche), for 3 h at 4°C with gentle agitation.

Immunoprecipitation:

Article Title: NF45 and NF90/NF110 coordinately regulate ESC pluripotency and differentiation
Article Snippet: ESCs were harvested, pelleted, and lysed in 10 pellet volumes of low NP40 lysis buffer (12.5 mM Tris pH 7.9, 150 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 10% glycerol) with cOmplete protease inhibitors (Roche). .. Antibodies (Ab) for immunoprecipitation (5 µg NF45 Ab/1 mL lysate [Everest Biotech EB07784], 5 µg NF90/NF110 Ab/1 mL lysate [BD Biosciences clone 21/DRBP76], 6 µg normal rabbit IgG/1 mL lysate [Thermo Fisher Scientific]) were conjugated to 40 µL Protein G Dynabeads/1 mL lysate (Thermo Fisher Scientific) in low NP40 lysis buffer for 2 h at room temperature.

Article Title: TTC5 is required to prevent apoptosis of acute myeloid leukemia stem cells
Article Snippet: Paragraph title: Western blotting and immunoprecipitation ... For co-immunoprecipitation, cells were lysed in NP40 Lysis Buffer (50 mM Tris/HCl (pH 8.0), 50 mM KCl, 10 mM EDTA, 1% NP40, 6.25 mM NaF, 20 mM β -glycerophosphate, 1 mM DTT and 1 × Protease Inhibitor cocktail (Roche)).

Protease Inhibitor:

Article Title: Contribution of SUMO-interacting motifs and SUMOylation to the antiretroviral properties of TRIM5?
Article Snippet: .. Cells were washed in phosphate-buffered saline (PBS) and lysed in NP40 lysis buffer (0.5% Nonidet P40 (NP40), 1X protease inhibitor (complete EDTA-free, Roche Diagnostics) in PBS) for 45 min at 4 °C. ..

Article Title: The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS
Article Snippet: .. Lysates were collected by scraping, transferred to microtubes and subjected to two rounds of boiling at 100°C for 5 min followed by sonication for 1 min. For crude fractionation experiments, we used NP40 lysis buffer containing 50 m m Tris–Cl, pH 7.5, 150 m m NaCl and 1% NP-40, supplemented with 1 m m DTT, 1 m m PMSF and Complete™ Protease Inhibitor Cocktail (Roche). ..

Article Title: BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum
Article Snippet: .. Immunoprecipitations, Pulse-Chase Experiments, and SDS-PAGE Cells were lysed in NP40 lysis buffer (0.5% NP40, 10 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2 , pH7.4) supplemented with a complete protease inhibitor cocktail (Roche) and 2.5 mM N-Ethylmaleimide. .. The immunoprecipiation was performed using 30 µl of immobilized rProtein A (IPA 300, Repligen) with the relevant antibodies, or with 12 µl anti-HA Affinity Matrix (3F10, Roche), for 3 h at 4°C with gentle agitation.

Article Title: Phosphorylation of MAVS/VISA by Nemo-like kinase (NLK) for degradation regulates the antiviral innate immune response
Article Snippet: .. Coimmunoprecipitation and immunoblot analyses HEK293T cells (1 × 106 ) were transfected or stimulated and harvested in 400 µL of NP40 lysis buffer (30 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1% NP40) with a protease inhibitor cocktail (Roche, Basel, Switzerland). .. Next, the supernatants were incubated with the indicated antibodies and Protein G beads (Roche) at 4 °C for 5 h. The beads were then washed three times with lysis buffer, and immunoprecipitants were eluted with SDS loading buffer and fractionated on SDS-PAGE gels.

Article Title: TTC5 is required to prevent apoptosis of acute myeloid leukemia stem cells
Article Snippet: .. For co-immunoprecipitation, cells were lysed in NP40 Lysis Buffer (50 mM Tris/HCl (pH 8.0), 50 mM KCl, 10 mM EDTA, 1% NP40, 6.25 mM NaF, 20 mM β -glycerophosphate, 1 mM DTT and 1 × Protease Inhibitor cocktail (Roche)). .. Lysate was incubated overnight with the appropriate antibody and immune complexes were pulled down with Protein G–Sepharose (Sigma).

Article Title: Synergistic Antitumor Effects of Endostar in Combination with Oxaliplatin via Inhibition of HIF and CXCR4 in the Colorectal Cell Line SW1116
Article Snippet: .. Western Blot Whole cell lysates from SW1116 were prepared in NP40 lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). ..

Article Title: miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs
Article Snippet: .. Western blot Cell lines and mouse pituitary tissues were lysed in NP40 lysis buffer: 250 mM NaCl, Tris 50 mM (pH 8.0), 5 mM EDTA, 0.5% NP-40 (v/v) and 2× Protease inhibitor tablets (Roche). .. Pituitary tissue samples were removed from −80°C storage immediately prior to use and homogenised in an appropriate volume of ice-cold NP40 lysis buffer.

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: .. The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche). .. In all cases, tissue lysates were electrophoresed under reducing conditions on gels ranging from 8% to 16%, or on 4% to 20% gradient gels (Mini-PROTEAN® TGX™ Precast Protein Gels, Bio-Rad, Inc., Hercules, CA), depending on the size of the examined protein.

Cell Culture:

Article Title: The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS
Article Snippet: Paragraph title: Protein extraction from cultured cells ... Lysates were collected by scraping, transferred to microtubes and subjected to two rounds of boiling at 100°C for 5 min followed by sonication for 1 min. For crude fractionation experiments, we used NP40 lysis buffer containing 50 m m Tris–Cl, pH 7.5, 150 m m NaCl and 1% NP-40, supplemented with 1 m m DTT, 1 m m PMSF and Complete™ Protease Inhibitor Cocktail (Roche).

Indirect Immunoperoxidase Assay:

Article Title: BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum
Article Snippet: Immunoprecipitations, Pulse-Chase Experiments, and SDS-PAGE Cells were lysed in NP40 lysis buffer (0.5% NP40, 10 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2 , pH7.4) supplemented with a complete protease inhibitor cocktail (Roche) and 2.5 mM N-Ethylmaleimide. .. The immunoprecipiation was performed using 30 µl of immobilized rProtein A (IPA 300, Repligen) with the relevant antibodies, or with 12 µl anti-HA Affinity Matrix (3F10, Roche), for 3 h at 4°C with gentle agitation.

Protein Concentration:

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: The pulverized tissue was lysed, as described for mass spectrometry analysis, normalized for equivalent protein concentration using a BCA Protein Assay Kit (Pierce–ThermoScientific, Rockford, IL) and samples from the indicated number of mice at the same age were pooled. .. The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche).

Sonication:

Article Title: The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS
Article Snippet: .. Lysates were collected by scraping, transferred to microtubes and subjected to two rounds of boiling at 100°C for 5 min followed by sonication for 1 min. For crude fractionation experiments, we used NP40 lysis buffer containing 50 m m Tris–Cl, pH 7.5, 150 m m NaCl and 1% NP-40, supplemented with 1 m m DTT, 1 m m PMSF and Complete™ Protease Inhibitor Cocktail (Roche). ..

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: .. The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche). .. In all cases, tissue lysates were electrophoresed under reducing conditions on gels ranging from 8% to 16%, or on 4% to 20% gradient gels (Mini-PROTEAN® TGX™ Precast Protein Gels, Bio-Rad, Inc., Hercules, CA), depending on the size of the examined protein.

Binding Assay:

Article Title: Synergistic Antitumor Effects of Endostar in Combination with Oxaliplatin via Inhibition of HIF and CXCR4 in the Colorectal Cell Line SW1116
Article Snippet: Western Blot Whole cell lysates from SW1116 were prepared in NP40 lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). .. After transfer, blocking of unspecific binding sites was achieved by incubation in TBST (50 mM Tris/HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.2) containing 5% skimmed milk.

Nucleic Acid Electrophoresis:

Article Title: miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs
Article Snippet: Western blot Cell lines and mouse pituitary tissues were lysed in NP40 lysis buffer: 250 mM NaCl, Tris 50 mM (pH 8.0), 5 mM EDTA, 0.5% NP-40 (v/v) and 2× Protease inhibitor tablets (Roche). .. Lysates were prepared in 5× Laemmli loading dye: 45 mmol/L Tris (pH 6.8), 10% glycerol, 1% SDS, 50 mmol/L DTT, 0.01% bromophenol blue, boiled at 95°C for 5 min and resolved using SDS-PAGE gel electrophoresis.

Labeling:

Article Title: BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum
Article Snippet: Immunoprecipitations, Pulse-Chase Experiments, and SDS-PAGE Cells were lysed in NP40 lysis buffer (0.5% NP40, 10 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2 , pH7.4) supplemented with a complete protease inhibitor cocktail (Roche) and 2.5 mM N-Ethylmaleimide. .. To achieve steady-state protein labeling, cells were incubated overnight with 500 µCi of [35 S]methionine/cysteine (Perkin Elmer) in methionine/cysteine-free DMEM supplemented with 10% dialyzed IFS at 37°C.

Mouse Assay:

Article Title: Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes
Article Snippet: Adipocytes that had been differentiated from the stromal vascular fractions of Angptl4 −/− and wild-type mice were prepared as described earlier. .. Upon differentiation, adipocytes were lysed in NP40 lysis buffer [50 mM Tris-HCl (pH 8.0), 0.5% NP40, 150 mM NaCl, 5 mM MgCl2 ] supplemented with protease and phosphatase inhibitors (Roche).

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: The pulverized tissue was lysed, as described for mass spectrometry analysis, normalized for equivalent protein concentration using a BCA Protein Assay Kit (Pierce–ThermoScientific, Rockford, IL) and samples from the indicated number of mice at the same age were pooled. .. The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche).

Dot Blot:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: Paragraph title: Transfection, cell lysates, Western blot analysis and dot blot assay ... At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

Protein Extraction:

Article Title: The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS
Article Snippet: Paragraph title: Protein extraction from cultured cells ... Lysates were collected by scraping, transferred to microtubes and subjected to two rounds of boiling at 100°C for 5 min followed by sonication for 1 min. For crude fractionation experiments, we used NP40 lysis buffer containing 50 m m Tris–Cl, pH 7.5, 150 m m NaCl and 1% NP-40, supplemented with 1 m m DTT, 1 m m PMSF and Complete™ Protease Inhibitor Cocktail (Roche).

Polyacrylamide Gel Electrophoresis:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Article Title: Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1? and TRIF/IRF3
Article Snippet: IRF3 Monocytes or macrophages were primed with 1 ng/ml of LPS or with 50 ng/ml of Pam3CSK4 for 1 h. At the end of incubation period, cells were lysed in 1% NP40 lysis buffer with PI, PMSF, and PhosSTOP Phosphatase Inhibitor (Roche Diagnostics, Indianapolis, IN) on ice for 30 min. .. Cell lysates were resolved using 7.5% Tris-Glycine Page Gold Precast gel (Lonza, Rockland, ME) and Tris-Glycine SDS running buffer.

SDS Page:

Article Title: Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes
Article Snippet: Upon differentiation, adipocytes were lysed in NP40 lysis buffer [50 mM Tris-HCl (pH 8.0), 0.5% NP40, 150 mM NaCl, 5 mM MgCl2 ] supplemented with protease and phosphatase inhibitors (Roche). .. NP40S and NP40P fractions were then loaded onto SDS-PAGE for further analyses by Western blotting.

Article Title: BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum
Article Snippet: .. Immunoprecipitations, Pulse-Chase Experiments, and SDS-PAGE Cells were lysed in NP40 lysis buffer (0.5% NP40, 10 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2 , pH7.4) supplemented with a complete protease inhibitor cocktail (Roche) and 2.5 mM N-Ethylmaleimide. .. The immunoprecipiation was performed using 30 µl of immobilized rProtein A (IPA 300, Repligen) with the relevant antibodies, or with 12 µl anti-HA Affinity Matrix (3F10, Roche), for 3 h at 4°C with gentle agitation.

Article Title: Phosphorylation of MAVS/VISA by Nemo-like kinase (NLK) for degradation regulates the antiviral innate immune response
Article Snippet: Coimmunoprecipitation and immunoblot analyses HEK293T cells (1 × 106 ) were transfected or stimulated and harvested in 400 µL of NP40 lysis buffer (30 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1% NP40) with a protease inhibitor cocktail (Roche, Basel, Switzerland). .. Next, the supernatants were incubated with the indicated antibodies and Protein G beads (Roche) at 4 °C for 5 h. The beads were then washed three times with lysis buffer, and immunoprecipitants were eluted with SDS loading buffer and fractionated on SDS-PAGE gels.

Article Title: TTC5 is required to prevent apoptosis of acute myeloid leukemia stem cells
Article Snippet: Western blotting and immunoprecipitation For western blotting, cells were lysed in High Salt Lysis Buffer (45 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 6.25 mM NaF, 20 mM β -glycerophosphate, 1 mM DTT, 20 mM sodium butyrate and 1 × Protease Inhibitor cocktail (Roche, Burgess Hill, UK)) and equal amounts of protein were loaded and separated by SDS-PAGE. .. For co-immunoprecipitation, cells were lysed in NP40 Lysis Buffer (50 mM Tris/HCl (pH 8.0), 50 mM KCl, 10 mM EDTA, 1% NP40, 6.25 mM NaF, 20 mM β -glycerophosphate, 1 mM DTT and 1 × Protease Inhibitor cocktail (Roche)).

Article Title: miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs
Article Snippet: Western blot Cell lines and mouse pituitary tissues were lysed in NP40 lysis buffer: 250 mM NaCl, Tris 50 mM (pH 8.0), 5 mM EDTA, 0.5% NP-40 (v/v) and 2× Protease inhibitor tablets (Roche). .. Lysates were prepared in 5× Laemmli loading dye: 45 mmol/L Tris (pH 6.8), 10% glycerol, 1% SDS, 50 mmol/L DTT, 0.01% bromophenol blue, boiled at 95°C for 5 min and resolved using SDS-PAGE gel electrophoresis.

Plasmid Preparation:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: Transfection, cell lysates, Western blot analysis and dot blot assay 293T cells (from ATCC) prepared in a 10 cm-culture dish at 5×105 cells per dish one day earlier were transfected with 10 µg of plasmid DNA by calcium phosphate method . .. At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

Concentration Assay:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Fractionation:

Article Title: The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS
Article Snippet: .. Lysates were collected by scraping, transferred to microtubes and subjected to two rounds of boiling at 100°C for 5 min followed by sonication for 1 min. For crude fractionation experiments, we used NP40 lysis buffer containing 50 m m Tris–Cl, pH 7.5, 150 m m NaCl and 1% NP-40, supplemented with 1 m m DTT, 1 m m PMSF and Complete™ Protease Inhibitor Cocktail (Roche). ..

Lysis:

Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins
Article Snippet: .. At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates . .. For Western blot analysis, cell lysates were added to non-reducing buffer (2% SDS, 0.5 M Tris [pH 6.8], 20% glycerol, 0.001% bromophenol blue [final concentration]) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membrane, blocking and incubation with primary antibody (human sera from confirmed dengue cases) and secondary antibody , .

Article Title: NF45 and NF90/NF110 coordinately regulate ESC pluripotency and differentiation
Article Snippet: .. ESCs were harvested, pelleted, and lysed in 10 pellet volumes of low NP40 lysis buffer (12.5 mM Tris pH 7.9, 150 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 10% glycerol) with cOmplete protease inhibitors (Roche). .. Lysates were freeze-thawed 3 times and then cleared by centrifugation at maximum speed for 15 min at 4°C.

Article Title: Contribution of SUMO-interacting motifs and SUMOylation to the antiretroviral properties of TRIM5?
Article Snippet: .. Cells were washed in phosphate-buffered saline (PBS) and lysed in NP40 lysis buffer (0.5% Nonidet P40 (NP40), 1X protease inhibitor (complete EDTA-free, Roche Diagnostics) in PBS) for 45 min at 4 °C. ..

Article Title: Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1? and TRIF/IRF3
Article Snippet: .. IRF3 Monocytes or macrophages were primed with 1 ng/ml of LPS or with 50 ng/ml of Pam3CSK4 for 1 h. At the end of incubation period, cells were lysed in 1% NP40 lysis buffer with PI, PMSF, and PhosSTOP Phosphatase Inhibitor (Roche Diagnostics, Indianapolis, IN) on ice for 30 min. .. Cell lysates were resolved using 7.5% Tris-Glycine Page Gold Precast gel (Lonza, Rockland, ME) and Tris-Glycine SDS running buffer.

Article Title: Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes
Article Snippet: .. Upon differentiation, adipocytes were lysed in NP40 lysis buffer [50 mM Tris-HCl (pH 8.0), 0.5% NP40, 150 mM NaCl, 5 mM MgCl2 ] supplemented with protease and phosphatase inhibitors (Roche). .. After centrifugation, the supernatant fluid (NP40S) was mixed with 2× Laemmli sample buffer and heated at 65°C for 15 min.

Article Title: The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS
Article Snippet: .. Lysates were collected by scraping, transferred to microtubes and subjected to two rounds of boiling at 100°C for 5 min followed by sonication for 1 min. For crude fractionation experiments, we used NP40 lysis buffer containing 50 m m Tris–Cl, pH 7.5, 150 m m NaCl and 1% NP-40, supplemented with 1 m m DTT, 1 m m PMSF and Complete™ Protease Inhibitor Cocktail (Roche). ..

Article Title: BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum
Article Snippet: .. Immunoprecipitations, Pulse-Chase Experiments, and SDS-PAGE Cells were lysed in NP40 lysis buffer (0.5% NP40, 10 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2 , pH7.4) supplemented with a complete protease inhibitor cocktail (Roche) and 2.5 mM N-Ethylmaleimide. .. The immunoprecipiation was performed using 30 µl of immobilized rProtein A (IPA 300, Repligen) with the relevant antibodies, or with 12 µl anti-HA Affinity Matrix (3F10, Roche), for 3 h at 4°C with gentle agitation.

Article Title: Phosphorylation of MAVS/VISA by Nemo-like kinase (NLK) for degradation regulates the antiviral innate immune response
Article Snippet: .. Coimmunoprecipitation and immunoblot analyses HEK293T cells (1 × 106 ) were transfected or stimulated and harvested in 400 µL of NP40 lysis buffer (30 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1% NP40) with a protease inhibitor cocktail (Roche, Basel, Switzerland). .. Next, the supernatants were incubated with the indicated antibodies and Protein G beads (Roche) at 4 °C for 5 h. The beads were then washed three times with lysis buffer, and immunoprecipitants were eluted with SDS loading buffer and fractionated on SDS-PAGE gels.

Article Title: TTC5 is required to prevent apoptosis of acute myeloid leukemia stem cells
Article Snippet: .. For co-immunoprecipitation, cells were lysed in NP40 Lysis Buffer (50 mM Tris/HCl (pH 8.0), 50 mM KCl, 10 mM EDTA, 1% NP40, 6.25 mM NaF, 20 mM β -glycerophosphate, 1 mM DTT and 1 × Protease Inhibitor cocktail (Roche)). .. Lysate was incubated overnight with the appropriate antibody and immune complexes were pulled down with Protein G–Sepharose (Sigma).

Article Title: Synergistic Antitumor Effects of Endostar in Combination with Oxaliplatin via Inhibition of HIF and CXCR4 in the Colorectal Cell Line SW1116
Article Snippet: .. Western Blot Whole cell lysates from SW1116 were prepared in NP40 lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). ..

Article Title: miR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs
Article Snippet: .. Western blot Cell lines and mouse pituitary tissues were lysed in NP40 lysis buffer: 250 mM NaCl, Tris 50 mM (pH 8.0), 5 mM EDTA, 0.5% NP-40 (v/v) and 2× Protease inhibitor tablets (Roche). .. Pituitary tissue samples were removed from −80°C storage immediately prior to use and homogenised in an appropriate volume of ice-cold NP40 lysis buffer.

Article Title: SIL1, the endoplasmic-reticulum-localized BiP co-chaperone, plays a crucial role in maintaining skeletal muscle proteostasis and physiology
Article Snippet: .. The NP40-insoluble pellets were washed once with NP40 lysis buffer and then solubilized in 50 mM Tris (pH 8) buffer, containing 0.6% SDS, sonicated for 4 min (30 s ON-30 s OFF cycles) and incubated at 95°C for 10 min. All buffers contained 0.25 mM phenylmethylsulphonyl fluoride (PMSF) and a protease inhibitor cocktail (Complete, Roche). .. In all cases, tissue lysates were electrophoresed under reducing conditions on gels ranging from 8% to 16%, or on 4% to 20% gradient gels (Mini-PROTEAN® TGX™ Precast Protein Gels, Bio-Rad, Inc., Hercules, CA), depending on the size of the examined protein.

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  • 96
    Roche np40 lysis buffer
    Focal accumulation of SIRT2 coincides with reduced MT acetylation in neurons. ( A ). SIRT2 antibodies label MTs in processes of cultured primary cortical neurons (DIV 10). Panels show SIRT2 (left), total α-tubulin (middle) and merged image (right) indicating overlapping staining of SIRT2 and α-tubulin. ( B and C ). Areas of high-level endogenous SIRT2 expression (arrows) overlap with reduced α-tubulin acetylation in perikarya (B) and processes (C) of cultured primary neurons (DIV 11). Panels show SIRT2 (left), acetylated α-tubulin (middle) and merged images. ( D ). Fractionation of proteins from cultured primary neurons shows that endogenous SIRT2.2 is found primarily in the insoluble pellet (P) fraction; in contrast, SIRT2.1 is found largely in the soluble (S) fraction. Samples from five independent cultures are shown. ( E ). Brain-enriched SIRT2 isoforms are associated with the insoluble fraction in transfected cells. Like SIRT2.2, a significant amount of SIRT2.3 is found in the <t>NP40-insoluble</t> (P) fraction of transiently transfected N2a cells. Endogenous SIRT2.1 in these cells is found almost exclusively in the soluble (S) fraction.
    Np40 Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
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    90G4 monoclonal antibody recognizes the CD321 antigen. (A) Expression profile in endothelial cell lines. Indicated endothelial cell lines were tested by staining with control (gray) or 90G4 antibodies (black). Data of FITC fluorescent intensity (FL1) indicated in histogram. Data presents three independent experiments. (B) Biochemical profiling of the molecular weight of the putative antigen. SVEC4-10 cells were surface-biotinylated with Sulfo-NHS-Biotin and lysed in <t>NP40</t> buffer, followed by immunoprecipitation with 90G4 or IgG2a, к control antibodies. After SDS-polyacrylamide gel electrophoresis (PAGE), the putative antigen was visualized by probing the streptavidin-HRP (Str-HRP) conjugate by chemiluminescence. The molecular weight of the detected protein was 35 kDa. (C) Identification of 90G4 antigen. Amino acid sequence of CD321 proteins were indicated with the two of underlined peptide sequences that are detected by LC-MS/MS analysis. (D) Specific immunoreactivity of 90G4 antibody against CD321. The expression vectors encoding CD321, CD322, or CD323 cDNA were transfected into CHO cells, which were then subjected to flow cytometry analysis. Data are presented in contour plot (top) or overlaid in histogram (bottom) of FITC signal intensity of sample (black) or control (gray).
    Np40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 98/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche selectin wash lysis buffer
    <t>E-selectin</t> ligands co-localize with Glg1 at the cell surface. (a) Total internal reflectance fluorescence (TIRF) microscopy at the critical angle using a spinning disc was performed on SUM159-M1a cells probed with recombinant E-selectin-Fc (green) and anti-Glg1 (red). Scale bar represents 5 μm. (b) Confocal Z-slice of M1a cells probed with E-selectin-Fc (green), anti-Glg1 (red) and Hoechst (blue). Scale bar represents 5 μm. Data representative of 3 independent experiments ( a , b ).
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    Focal accumulation of SIRT2 coincides with reduced MT acetylation in neurons. ( A ). SIRT2 antibodies label MTs in processes of cultured primary cortical neurons (DIV 10). Panels show SIRT2 (left), total α-tubulin (middle) and merged image (right) indicating overlapping staining of SIRT2 and α-tubulin. ( B and C ). Areas of high-level endogenous SIRT2 expression (arrows) overlap with reduced α-tubulin acetylation in perikarya (B) and processes (C) of cultured primary neurons (DIV 11). Panels show SIRT2 (left), acetylated α-tubulin (middle) and merged images. ( D ). Fractionation of proteins from cultured primary neurons shows that endogenous SIRT2.2 is found primarily in the insoluble pellet (P) fraction; in contrast, SIRT2.1 is found largely in the soluble (S) fraction. Samples from five independent cultures are shown. ( E ). Brain-enriched SIRT2 isoforms are associated with the insoluble fraction in transfected cells. Like SIRT2.2, a significant amount of SIRT2.3 is found in the NP40-insoluble (P) fraction of transiently transfected N2a cells. Endogenous SIRT2.1 in these cells is found almost exclusively in the soluble (S) fraction.

    Journal: Human Molecular Genetics

    Article Title: The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS

    doi: 10.1093/hmg/ddr326

    Figure Lengend Snippet: Focal accumulation of SIRT2 coincides with reduced MT acetylation in neurons. ( A ). SIRT2 antibodies label MTs in processes of cultured primary cortical neurons (DIV 10). Panels show SIRT2 (left), total α-tubulin (middle) and merged image (right) indicating overlapping staining of SIRT2 and α-tubulin. ( B and C ). Areas of high-level endogenous SIRT2 expression (arrows) overlap with reduced α-tubulin acetylation in perikarya (B) and processes (C) of cultured primary neurons (DIV 11). Panels show SIRT2 (left), acetylated α-tubulin (middle) and merged images. ( D ). Fractionation of proteins from cultured primary neurons shows that endogenous SIRT2.2 is found primarily in the insoluble pellet (P) fraction; in contrast, SIRT2.1 is found largely in the soluble (S) fraction. Samples from five independent cultures are shown. ( E ). Brain-enriched SIRT2 isoforms are associated with the insoluble fraction in transfected cells. Like SIRT2.2, a significant amount of SIRT2.3 is found in the NP40-insoluble (P) fraction of transiently transfected N2a cells. Endogenous SIRT2.1 in these cells is found almost exclusively in the soluble (S) fraction.

    Article Snippet: Lysates were collected by scraping, transferred to microtubes and subjected to two rounds of boiling at 100°C for 5 min followed by sonication for 1 min. For crude fractionation experiments, we used NP40 lysis buffer containing 50 m m Tris–Cl, pH 7.5, 150 m m NaCl and 1% NP-40, supplemented with 1 m m DTT, 1 m m PMSF and Complete™ Protease Inhibitor Cocktail (Roche).

    Techniques: Cell Culture, Staining, Expressing, Fractionation, Transfection

    Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.

    Journal: PLoS ONE

    Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

    doi: 10.1371/journal.pone.0052600

    Figure Lengend Snippet: Effect of C-terminal E domains and prM protein on the recognition of E protein by different mouse anti-E mAbs. (A) Binding specificity of five mouse anti-E mAbs including GR (4G2 and DEN2-12), CR (DEN3-3), and DENV4 TS (1H10-6-7 and 1H10-5-7) mAbs. Western blot analysis was performed by using cell lysates derived from C6/36 cells infected with each of the four DENV serotypes, WNV or JEV. The size of molecular weight markers is shown in kDa. (B,C) Dot blot binding assay using these five mAbs to recognize WT E protein (expressed by prME), E protein alone and mutant E proteins containing C-terminal truncations (expressed by prME- or E-based constructs) in 1% NP40 lysis buffer (NP40). Layout of the dot blot assay and the binding by mixed mAbs are shown in (B). Decreasing amount of native WT E protein in 1% NP40 lysis buffer (column B) as well as mixtures containing decreasing amount of native WT E protein in 1% NP40 lysis buffer and increasing amount of denatured WT E protein in reducing (R) buffer (column A) were also included to control for exposure and sensitivity of the assay signal. Relative intensities of each dot in columns A (black bars) and B (white bars) were shown below each membrane. Recognition indices of each mAb to mutant E protein = [intensity of mutant E dot/intensity of WT E dot] (recognized by a mAb) divided by [intensity of mutant E dot/intensity of WT E dot] (recognized by mixed mAbs) were shown in blue bars (column C, in the presence of prM protein) and yellow bars (column D, in the absence of prM protein) below each membrane [33] . Data are mean and standard errors from two experiments.

    Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

    Techniques: Binding Assay, Western Blot, Derivative Assay, Infection, Molecular Weight, Dot Blot, Mutagenesis, Construct, Lysis

    Effect of E protein on the recognition of prM protein by human anti-prM mAbs. (A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5 . (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.

    Journal: PLoS ONE

    Article Title: C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

    doi: 10.1371/journal.pone.0052600

    Figure Lengend Snippet: Effect of E protein on the recognition of prM protein by human anti-prM mAbs. (A) Binding specificity of 4 human anti-prM mAbs including 2 CR (DVB59.3 and DVB18.5) and 2 sCR (DVB65.5 and DVB32.4) mAbs was determined as in Figure 5 . (B) Dot blot binding assay using these 4 mAbs to recognize prM protein in the presence (expressed by prME) or absence (expressed by prM) of E protein in 1% NP40 lysis buffer (NP40). Decreasing amount of prM protein (expressed by prME) in 1% NP40 lysis buffer (column B, rows 3 to 7) and mixtures containing decreasing amount of prM protein in 1% NP40 lysis buffer and increasing amount of denatured prM protein in reducing (R) buffer (column A) were included to control for exposure and sensitivity of the assay signal, respectively. Twenty times more cell lysates derived from transfection of prM alone were loaded. Relative intensities of each dot in column A (black bars) and column B (blue bars) were shown below each membrane. Data are mean and standard errors from two experiments.

    Article Snippet: At 48 h post-transfection, cells were washed with 1× PBS and treated with 1% NP40 lysis buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.5% Na deoxycholate and protease inhibitors [Roche Diagnostics]), followed by centrifugation at 20,000×g and 4°C for 30 min to obtain cell lysates .

    Techniques: Binding Assay, Dot Blot, Lysis, Derivative Assay, Transfection

    90G4 monoclonal antibody recognizes the CD321 antigen. (A) Expression profile in endothelial cell lines. Indicated endothelial cell lines were tested by staining with control (gray) or 90G4 antibodies (black). Data of FITC fluorescent intensity (FL1) indicated in histogram. Data presents three independent experiments. (B) Biochemical profiling of the molecular weight of the putative antigen. SVEC4-10 cells were surface-biotinylated with Sulfo-NHS-Biotin and lysed in NP40 buffer, followed by immunoprecipitation with 90G4 or IgG2a, к control antibodies. After SDS-polyacrylamide gel electrophoresis (PAGE), the putative antigen was visualized by probing the streptavidin-HRP (Str-HRP) conjugate by chemiluminescence. The molecular weight of the detected protein was 35 kDa. (C) Identification of 90G4 antigen. Amino acid sequence of CD321 proteins were indicated with the two of underlined peptide sequences that are detected by LC-MS/MS analysis. (D) Specific immunoreactivity of 90G4 antibody against CD321. The expression vectors encoding CD321, CD322, or CD323 cDNA were transfected into CHO cells, which were then subjected to flow cytometry analysis. Data are presented in contour plot (top) or overlaid in histogram (bottom) of FITC signal intensity of sample (black) or control (gray).

    Journal: PLoS ONE

    Article Title: A novel immunotoxin reveals a new role for CD321 in endothelial cells

    doi: 10.1371/journal.pone.0181502

    Figure Lengend Snippet: 90G4 monoclonal antibody recognizes the CD321 antigen. (A) Expression profile in endothelial cell lines. Indicated endothelial cell lines were tested by staining with control (gray) or 90G4 antibodies (black). Data of FITC fluorescent intensity (FL1) indicated in histogram. Data presents three independent experiments. (B) Biochemical profiling of the molecular weight of the putative antigen. SVEC4-10 cells were surface-biotinylated with Sulfo-NHS-Biotin and lysed in NP40 buffer, followed by immunoprecipitation with 90G4 or IgG2a, к control antibodies. After SDS-polyacrylamide gel electrophoresis (PAGE), the putative antigen was visualized by probing the streptavidin-HRP (Str-HRP) conjugate by chemiluminescence. The molecular weight of the detected protein was 35 kDa. (C) Identification of 90G4 antigen. Amino acid sequence of CD321 proteins were indicated with the two of underlined peptide sequences that are detected by LC-MS/MS analysis. (D) Specific immunoreactivity of 90G4 antibody against CD321. The expression vectors encoding CD321, CD322, or CD323 cDNA were transfected into CHO cells, which were then subjected to flow cytometry analysis. Data are presented in contour plot (top) or overlaid in histogram (bottom) of FITC signal intensity of sample (black) or control (gray).

    Article Snippet: To stop the reaction, cells were treated with PBS (-) containing 100 mM glycine, followed by lysis in NP40 buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.6, 1% NP40, and complete Roche protease inhibitor cocktail).

    Techniques: Expressing, Staining, Molecular Weight, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transfection, Flow Cytometry, Cytometry

    E-selectin ligands co-localize with Glg1 at the cell surface. (a) Total internal reflectance fluorescence (TIRF) microscopy at the critical angle using a spinning disc was performed on SUM159-M1a cells probed with recombinant E-selectin-Fc (green) and anti-Glg1 (red). Scale bar represents 5 μm. (b) Confocal Z-slice of M1a cells probed with E-selectin-Fc (green), anti-Glg1 (red) and Hoechst (blue). Scale bar represents 5 μm. Data representative of 3 independent experiments ( a , b ).

    Journal: Nature cell biology

    Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis

    doi: 10.1038/s41556-019-0309-2

    Figure Lengend Snippet: E-selectin ligands co-localize with Glg1 at the cell surface. (a) Total internal reflectance fluorescence (TIRF) microscopy at the critical angle using a spinning disc was performed on SUM159-M1a cells probed with recombinant E-selectin-Fc (green) and anti-Glg1 (red). Scale bar represents 5 μm. (b) Confocal Z-slice of M1a cells probed with E-selectin-Fc (green), anti-Glg1 (red) and Hoechst (blue). Scale bar represents 5 μm. Data representative of 3 independent experiments ( a , b ).

    Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)).

    Techniques: Fluorescence, Microscopy, Recombinant

    E-selectin-induced MET activates Wnt signaling. (a) BM2 cells stably expressing the 7x TCF-GFP reporter and SV40-mCherry as internal control (BM2-TGC) were plated on either E-selectin or IgG coated (10 μg/mL) plates with or without recombinant Wnt3a (100 ng/mL). Fluorescence was assessed by confocal microscopy after 48 h and quantified by flow cytometry. Scale bars represent 100 μm. Data representative of > 5 independent experiments. (b) qPCR analysis of EMT- or Wnt-associated genes from the BM2 cells plated on E-selectin or IgG (10 μg/mL) for 48 h. n= 3 technical replicates. (c) Ex vivo confocal images of bone metastasis in Nu/Nu mice injected with BM2-TGC. Live animal labeling with anti-CD31 was conducted to visualize vasculature. Scale bar represents 100 μm. Data representative of 4 biological replicates. (d) qPCR of Glg1-variant 3 , Fut3 and Fut6 levels in BM2 cells seeded on either IgG or E-selectin (10 μg/mL) for 48 h. n = 4 technical replicates. (e) BM2 cells stably-expressing the SORE6-mCherry stemness reporter or control mCherry reporter were plated on E-selectin or control IgG (10 μg/mL) for 48 h. mCherry fluorescence was assessed by flow cytometry after 48 h. Data representative of 3 independent biological replicates. (f) Proposed model of E-selectin function during bone metastasis. Data represent mean ± SEM.

    Journal: Nature cell biology

    Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis

    doi: 10.1038/s41556-019-0309-2

    Figure Lengend Snippet: E-selectin-induced MET activates Wnt signaling. (a) BM2 cells stably expressing the 7x TCF-GFP reporter and SV40-mCherry as internal control (BM2-TGC) were plated on either E-selectin or IgG coated (10 μg/mL) plates with or without recombinant Wnt3a (100 ng/mL). Fluorescence was assessed by confocal microscopy after 48 h and quantified by flow cytometry. Scale bars represent 100 μm. Data representative of > 5 independent experiments. (b) qPCR analysis of EMT- or Wnt-associated genes from the BM2 cells plated on E-selectin or IgG (10 μg/mL) for 48 h. n= 3 technical replicates. (c) Ex vivo confocal images of bone metastasis in Nu/Nu mice injected with BM2-TGC. Live animal labeling with anti-CD31 was conducted to visualize vasculature. Scale bar represents 100 μm. Data representative of 4 biological replicates. (d) qPCR of Glg1-variant 3 , Fut3 and Fut6 levels in BM2 cells seeded on either IgG or E-selectin (10 μg/mL) for 48 h. n = 4 technical replicates. (e) BM2 cells stably-expressing the SORE6-mCherry stemness reporter or control mCherry reporter were plated on E-selectin or control IgG (10 μg/mL) for 48 h. mCherry fluorescence was assessed by flow cytometry after 48 h. Data representative of 3 independent biological replicates. (f) Proposed model of E-selectin function during bone metastasis. Data represent mean ± SEM.

    Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)).

    Techniques: Stable Transfection, Expressing, Recombinant, Fluorescence, Confocal Microscopy, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Ex Vivo, Mouse Assay, Injection, Labeling, Variant Assay

    E-selectin binding to tumor cells induces a non-canonical mesenchymal-to-epithelial transition. (a) Confocal imaging of E-selectin-induced morphological changes in M1a cells after culturing on E-selectin- compared to IgG-coated plates (10 μg/mL) for 24 h. Z-slice of confocal immunofluorescent images demonstrating N-cadherin and Tight Junction Protein-1 (ZO-1) localization/expression changes in M1a cells. Scale bars represent 20 μm. ( b) Confocal imaging of EpCam immunofluorescence in BM2 cells seeded over E-selectin or IgG coated plates. Scale bars represent 20 μm. Images representative of 3 independent experiments (a–b). (c) GSEA of the Sarrio EMT Down (GSE8430), Hallmark EMT Up (M5930), and Claudin-low Up 61 gene sets in the ranked gene list of the BM2 and M1a cells cultured in E-selectin- vs. IgG-coated plates. Single mRNA isolation used for each condition in each cell line. p and q statistics by GSEA software, n = 108 gene sets queried. (d) Heat map of the expression of known EMT transcriptional regulators in BM2 and M1a cells cultured in E-selectin vs. IgG coated plates. (e) Overlapping enrichment core genes from the Sarrio EMT down gene set (positive) and Hallmark EMT Up gene set (negative) between the BM2 and M1a cells were compiled to form the E-selectin-induced MET gene signature shown in the heatmap. The gene identities are listed in Supplementary Table 5 . (f) qPCR analysis of Dkk1 and Ctgf mRNA levels in BM2 and M1a cells seeded over IgG or E-selectin for 24h. n = 3 technical replicates. Data represent mean ± SEM.

    Journal: Nature cell biology

    Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis

    doi: 10.1038/s41556-019-0309-2

    Figure Lengend Snippet: E-selectin binding to tumor cells induces a non-canonical mesenchymal-to-epithelial transition. (a) Confocal imaging of E-selectin-induced morphological changes in M1a cells after culturing on E-selectin- compared to IgG-coated plates (10 μg/mL) for 24 h. Z-slice of confocal immunofluorescent images demonstrating N-cadherin and Tight Junction Protein-1 (ZO-1) localization/expression changes in M1a cells. Scale bars represent 20 μm. ( b) Confocal imaging of EpCam immunofluorescence in BM2 cells seeded over E-selectin or IgG coated plates. Scale bars represent 20 μm. Images representative of 3 independent experiments (a–b). (c) GSEA of the Sarrio EMT Down (GSE8430), Hallmark EMT Up (M5930), and Claudin-low Up 61 gene sets in the ranked gene list of the BM2 and M1a cells cultured in E-selectin- vs. IgG-coated plates. Single mRNA isolation used for each condition in each cell line. p and q statistics by GSEA software, n = 108 gene sets queried. (d) Heat map of the expression of known EMT transcriptional regulators in BM2 and M1a cells cultured in E-selectin vs. IgG coated plates. (e) Overlapping enrichment core genes from the Sarrio EMT down gene set (positive) and Hallmark EMT Up gene set (negative) between the BM2 and M1a cells were compiled to form the E-selectin-induced MET gene signature shown in the heatmap. The gene identities are listed in Supplementary Table 5 . (f) qPCR analysis of Dkk1 and Ctgf mRNA levels in BM2 and M1a cells seeded over IgG or E-selectin for 24h. n = 3 technical replicates. Data represent mean ± SEM.

    Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)).

    Techniques: Binding Assay, Imaging, Expressing, Immunofluorescence, Cell Culture, Isolation, Software, Real-time Polymerase Chain Reaction

    Specific α1–3 Fucosyltransferases (Fut3 and Fut6) promote bone metastasis. (a) Comparative flow cytometry analysis of E-selectin binding to MDA-MB-231 cells with stable ectopic expression of α1–3 Fut enzymes. MDA-MB-231-RFP was used as an internal control. Data representative of four independent experiments. (b) BLI quantification of bone metastasis burden following intracardiac injection of M1a cells stably expressing each Fut enzyme into Nu/Nu mice. n = 6 (Fut3, Fut4, Fut6), 5 (Fut5, Fut9), 3 (Fut7), and 10 (Vector) mice. Statistics by Mann-Whitney U test at Day 21, two-sided. (c) Representative BLI and X-ray images of bone lesions from (b). White arrows indicate osteolytic lesions. (d) qPCR analysis of endogenous α1–3 Fut mRNA levels in the parental and sorted MDA-MB-231 cells with differential E-selectin binding abilities. Fut5/7/9 were not detectable (N.D.) in all cell lines. n = 3 technical replicates. (e) Tumor volume measurements after orthotopic injection of M1a cells stably-expressing each relevant Fut enzyme into NSG mice. p > 0.05 for all between-group comparisons by two-sided Student’s t-test at Day 39. n = 6 mice/group. (f,g) Violin plot showing BLI quantification of spontaneous bone (f) and lung (g) metastasis burden. Plot elements include median, box for interquartile range, spikes to upper- and lower- adjacent values. Statistics by Mann Whitney U test, one-sided. n = 11 lung/22 hindlimb (Fut3 and Fut 4), 6 lung/12 hindlimb (Fut 6 and Fut 7), and 12 lung/24 hindlimb (Vector). (h) Representative BLI images of bone and lung tissues from each experimental group. Experiment performed once (e-h). Data represent mean ± SEM. No statistically significant difference (p > 0.05) between Fut4, Fut7, and Fut9 groups vs Vector group in b , Fut 4 and Fut 7 groups vs Vector group in f , and any group vs Vector group in e and g .

    Journal: Nature cell biology

    Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis

    doi: 10.1038/s41556-019-0309-2

    Figure Lengend Snippet: Specific α1–3 Fucosyltransferases (Fut3 and Fut6) promote bone metastasis. (a) Comparative flow cytometry analysis of E-selectin binding to MDA-MB-231 cells with stable ectopic expression of α1–3 Fut enzymes. MDA-MB-231-RFP was used as an internal control. Data representative of four independent experiments. (b) BLI quantification of bone metastasis burden following intracardiac injection of M1a cells stably expressing each Fut enzyme into Nu/Nu mice. n = 6 (Fut3, Fut4, Fut6), 5 (Fut5, Fut9), 3 (Fut7), and 10 (Vector) mice. Statistics by Mann-Whitney U test at Day 21, two-sided. (c) Representative BLI and X-ray images of bone lesions from (b). White arrows indicate osteolytic lesions. (d) qPCR analysis of endogenous α1–3 Fut mRNA levels in the parental and sorted MDA-MB-231 cells with differential E-selectin binding abilities. Fut5/7/9 were not detectable (N.D.) in all cell lines. n = 3 technical replicates. (e) Tumor volume measurements after orthotopic injection of M1a cells stably-expressing each relevant Fut enzyme into NSG mice. p > 0.05 for all between-group comparisons by two-sided Student’s t-test at Day 39. n = 6 mice/group. (f,g) Violin plot showing BLI quantification of spontaneous bone (f) and lung (g) metastasis burden. Plot elements include median, box for interquartile range, spikes to upper- and lower- adjacent values. Statistics by Mann Whitney U test, one-sided. n = 11 lung/22 hindlimb (Fut3 and Fut 4), 6 lung/12 hindlimb (Fut 6 and Fut 7), and 12 lung/24 hindlimb (Vector). (h) Representative BLI images of bone and lung tissues from each experimental group. Experiment performed once (e-h). Data represent mean ± SEM. No statistically significant difference (p > 0.05) between Fut4, Fut7, and Fut9 groups vs Vector group in b , Fut 4 and Fut 7 groups vs Vector group in f , and any group vs Vector group in e and g .

    Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)).

    Techniques: Flow Cytometry, Cytometry, Binding Assay, Multiple Displacement Amplification, Expressing, Injection, Stable Transfection, Mouse Assay, Plasmid Preparation, MANN-WHITNEY, Real-time Polymerase Chain Reaction

    In vitro and in vivo characterization of Fut3 catalytic mutants. (a) Western blot detection of three Fut3 catalytic mutants compared to wild-type Fut3 ectopically expressed in SUM159-M1a cells. (b) Comparative flow cytometry analysis of E-selectin binding to M1a cells expressing each Fut3 mutant using SUM159-RFP as an internal control. Data representative of 3 independent experiments (a,b). (c) BLI quantification of bone metastasis burden following intracardiac injection of M1a cells stably expressing each Fut3 mutant compared to vector and wild-type Fut3. n = 8 (Fut3 and Y315-stop) and 9 (E247K and Vector) mice/group. Mann-Whitney U test, two-sided. (d) Representative BLI and X-ray images from (c). (e,f) Quantification of osteloytic area (e) and the number of osteolytic lesions (f) in the hind limbs of Nu/Nu mice receiving an intracardiac injection of M1a cells expressing each Fut3 mutant. n = 16 (Fut3 and Y315-stop) and 18 (E247K and Vector) hindlimbs/group. Mann-Whitney U test, two-sided. Experiment performed once ( c – f ). Data represent mean ± SEM. Unprocessed original scans of the blots in a are shown Supplementary Fig. 9 .

    Journal: Nature cell biology

    Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis

    doi: 10.1038/s41556-019-0309-2

    Figure Lengend Snippet: In vitro and in vivo characterization of Fut3 catalytic mutants. (a) Western blot detection of three Fut3 catalytic mutants compared to wild-type Fut3 ectopically expressed in SUM159-M1a cells. (b) Comparative flow cytometry analysis of E-selectin binding to M1a cells expressing each Fut3 mutant using SUM159-RFP as an internal control. Data representative of 3 independent experiments (a,b). (c) BLI quantification of bone metastasis burden following intracardiac injection of M1a cells stably expressing each Fut3 mutant compared to vector and wild-type Fut3. n = 8 (Fut3 and Y315-stop) and 9 (E247K and Vector) mice/group. Mann-Whitney U test, two-sided. (d) Representative BLI and X-ray images from (c). (e,f) Quantification of osteloytic area (e) and the number of osteolytic lesions (f) in the hind limbs of Nu/Nu mice receiving an intracardiac injection of M1a cells expressing each Fut3 mutant. n = 16 (Fut3 and Y315-stop) and 18 (E247K and Vector) hindlimbs/group. Mann-Whitney U test, two-sided. Experiment performed once ( c – f ). Data represent mean ± SEM. Unprocessed original scans of the blots in a are shown Supplementary Fig. 9 .

    Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)).

    Techniques: In Vitro, In Vivo, Western Blot, Flow Cytometry, Cytometry, Binding Assay, Expressing, Mutagenesis, Injection, Stable Transfection, Plasmid Preparation, Mouse Assay, MANN-WHITNEY

    Cell surface N-glycan analysis reveals candidate E-selectin ligands in metastatic breast cancer cells. (a) Summary of mass spectrometry results of N-glycosites and N-glyco proteins using two biological replicates of each cell line collected over independent isolations. (b) Western blot confirming CD44 knockout by CRISPR-Cas9 in the BM2 cell line. Data representative of 3 independent experiments. (c) Comparative flow cytometry analysis of E-selectin binding to CD44-KO and control BM2 cell lines. MDA-MB-231-RFP used as an internal control and binding levels normalized to Ctrl-1. n = 4 independent biological replicates, Student’s t-test, two-sided. (d) Representative fragmentation spectra of the doubly-charged Glg1 tryptic peptide LNLTTDPK containing deamidated Asn(N)165 (red). N- and C-terminal (b and y) ions matching the predicted peptide fragment are indicated with their respective mass-to-charge (m/z) values. A complete y ion series was observed, including the site-determining y7 ion, localizing the deamidation to the Asn residue. The measured doubly-charged monoisotopic precursor ion m/z and its mass deviation (Δm) relative to the theoretical m/z are indicated. * interfering m/z from lower abundance co-isolated ion(s). Similar fragmentation spectra were obtained across independent isolations. (e) Schematic diagram of the three major Glg1 splice variants in humans. (f) Western blot analysis of Glg1 expression in SUM159 and M1a cells after stable transduction of retroviral vector expressing Glg1 variant 3. (g) Western blot analysis of Glg1 and CD44 after population-level CRISPR/Cas9 knockout of Glg1 and CD44 in M1a cells. Data representative of three independent experiments ( f , g ). (h) Comparative flow cytometry analysis of E-selectin binding to genetically manipulated SUM159 and M1a cells using SUM159-RFP as an internal control. Binding levels were normalized to respective controls. n = 5 (Glg1 or Vector), n = 3 (KO cell lines) independent biological replicates. Student’s t-test, two-sided, compares Glg1v3 cells to Vector cells and CRISPR-KO cells to CRISPR-Control cells. Data represent mean ± SEM. Unprocessed original scans of the blots in b , f , g are shown Supplementary Fig. 9 .

    Journal: Nature cell biology

    Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis

    doi: 10.1038/s41556-019-0309-2

    Figure Lengend Snippet: Cell surface N-glycan analysis reveals candidate E-selectin ligands in metastatic breast cancer cells. (a) Summary of mass spectrometry results of N-glycosites and N-glyco proteins using two biological replicates of each cell line collected over independent isolations. (b) Western blot confirming CD44 knockout by CRISPR-Cas9 in the BM2 cell line. Data representative of 3 independent experiments. (c) Comparative flow cytometry analysis of E-selectin binding to CD44-KO and control BM2 cell lines. MDA-MB-231-RFP used as an internal control and binding levels normalized to Ctrl-1. n = 4 independent biological replicates, Student’s t-test, two-sided. (d) Representative fragmentation spectra of the doubly-charged Glg1 tryptic peptide LNLTTDPK containing deamidated Asn(N)165 (red). N- and C-terminal (b and y) ions matching the predicted peptide fragment are indicated with their respective mass-to-charge (m/z) values. A complete y ion series was observed, including the site-determining y7 ion, localizing the deamidation to the Asn residue. The measured doubly-charged monoisotopic precursor ion m/z and its mass deviation (Δm) relative to the theoretical m/z are indicated. * interfering m/z from lower abundance co-isolated ion(s). Similar fragmentation spectra were obtained across independent isolations. (e) Schematic diagram of the three major Glg1 splice variants in humans. (f) Western blot analysis of Glg1 expression in SUM159 and M1a cells after stable transduction of retroviral vector expressing Glg1 variant 3. (g) Western blot analysis of Glg1 and CD44 after population-level CRISPR/Cas9 knockout of Glg1 and CD44 in M1a cells. Data representative of three independent experiments ( f , g ). (h) Comparative flow cytometry analysis of E-selectin binding to genetically manipulated SUM159 and M1a cells using SUM159-RFP as an internal control. Binding levels were normalized to respective controls. n = 5 (Glg1 or Vector), n = 3 (KO cell lines) independent biological replicates. Student’s t-test, two-sided, compares Glg1v3 cells to Vector cells and CRISPR-KO cells to CRISPR-Control cells. Data represent mean ± SEM. Unprocessed original scans of the blots in b , f , g are shown Supplementary Fig. 9 .

    Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)).

    Techniques: Mass Spectrometry, Western Blot, Knock-Out, CRISPR, Flow Cytometry, Cytometry, Binding Assay, Multiple Displacement Amplification, Isolation, Expressing, Transduction, Plasmid Preparation, Variant Assay

    E-selectin is critical for bone but not lung metastasis. (a,b) Bone, lung, and liver sections from WT or Sele −/− mice were assessed for E-selectin expression and CD31 co-localization by immunofluorescence. Scale bars: 100 μm. Data representative of three independent experiments. (c) BLI quantification of bone metastasis burden following intracardiac injection of the BM2 cell line into WT or Sele −/− SCID mice. n = 12 mice/group, Mann-Whitney U test, two-sided. (d) Representative BLI, X-ray, and μCT images of bone metastasis in WT and Sele −/− SCID mice. Images representative of median signal from (c). White arrows indicate osteolytic lesions. (e) Quantification of osteloytic area and the number of osteolytic lesions in the hind limbs of animals from (c). n = 23 Hindlimbs (WT), n = 24 hindlimbs (KO), Mann-Whitney U test, two-sided. Data representative of two independent experiments ( c , d , e ). Data represent mean ± SEM.

    Journal: Nature cell biology

    Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis

    doi: 10.1038/s41556-019-0309-2

    Figure Lengend Snippet: E-selectin is critical for bone but not lung metastasis. (a,b) Bone, lung, and liver sections from WT or Sele −/− mice were assessed for E-selectin expression and CD31 co-localization by immunofluorescence. Scale bars: 100 μm. Data representative of three independent experiments. (c) BLI quantification of bone metastasis burden following intracardiac injection of the BM2 cell line into WT or Sele −/− SCID mice. n = 12 mice/group, Mann-Whitney U test, two-sided. (d) Representative BLI, X-ray, and μCT images of bone metastasis in WT and Sele −/− SCID mice. Images representative of median signal from (c). White arrows indicate osteolytic lesions. (e) Quantification of osteloytic area and the number of osteolytic lesions in the hind limbs of animals from (c). n = 23 Hindlimbs (WT), n = 24 hindlimbs (KO), Mann-Whitney U test, two-sided. Data representative of two independent experiments ( c , d , e ). Data represent mean ± SEM.

    Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)).

    Techniques: Mouse Assay, Expressing, Immunofluorescence, Injection, MANN-WHITNEY