Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: (A, A′, A″) UAS Arm S10 is overexpressed in the dppGAL4 domain, which drives expression in a stripe at the A/P boundary. Red, green, and blue channels; representing (A) endogenous Armadillo, (A′) E-Cadherin-GFP under a ubiquitous promoter, and (A″) Arm S10 , respectively; are assessed separately from the same confocal section, here at the level of the AJ (60×2 magnification). The coloured lines through the images represent the cross-section at which intensity levels were measured. (B, B′, B″) Using NIH ImageJ software, a histogram is produced in which pixel intensity for each pixel is calculated across the confocal section for each channel. Median values are calculated from both wild type tissue (μ1) and the expression domains (μ2). (B, B′) μ1 is used as the baseline value for endogenous protein levels, and is used to set the proportion of protein in the AJ at p1 = 1. The proportion p2 of junctional protein in the expression domain is then calculated as the median value μ2/μ1 and is a fraction of p1. (B″) p1 is set to 0 as no protein is expected outside of the expression domain, while p2 is set to 1 as it is assumed that the maximal amount of Arm S10 will reside in the junction within the expression domain. This allows a distinction between zones of high and low expression levels, the latter being a fraction of p2, such that changes in endogenous protein levels can be monitored (not shown).

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Expressing, Software, Produced

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: (A, A′, A″) UAS Myr-ΔNArm 1–155 is overexpressed in the dppGAL4 domain, which drives expression in a stripe at the A/P boundary. Red, green, and blue channels; representing (A) endogenous Armadillo, (A′) E-Cadherin-GFP under a ubiquitous promoter, and (A″) Myr-ΔNArm 1–155 , respectively; are assessed separately from the same confocal section, here through the cytoplasm approximately 10% below the AJ (60×2 magnification). The red, green and blue spots represent the 10 data points selected from which to calculate median levels within the domain of expression (B, B′, B″). The white spots highlight the data points outside the domain of expression used to remove “background noise”, as the nuclei are expected to have zero pixel intensity here. Thus for each channel, p1 is calculated as μ2 subtracted from μ1, and normalised to a maximal pixel intensity of 255.

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: Armadillo-GFP (ArmGFP) and E-CadherinGFP (E-CadGFP) are expressed under endogenous Armadillo ubiquitous promoters, respectively. (A–C) Distribution of ArmGFP (A) and E-CadGFP (B) at AJs (red channels) are coincident; Wingless (Wg) and Patched (Ptc) outline the D/V and A/P boundaries, respectively, which are characterised by aligned cells (arrowheads, blue channel). (C) Mitotic cells express ArmGFP at the AJs, but lack septate junction markers immediately basally (red arrows, Scribble Scrb shown). (D) E-Cadherin (E-Cad, blue) and ArmGFP colocalise at membranes and in puncta (red, blue and white oultines). (E) Fasciclin III (FasIII, blue) crisply and exclusively marks basolateral membranes, with which ArmGFP puncta are closely associated (compare red and blue channels). The downregulation of FasIII at the A/P boundary (blue arrowheads) clearly outlines the ArmGFP puncta there (outlined in red).

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques:

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: (A, B) At 0%, Wingless signalling changes neither the levels nor the subcellular distribution of apicobasal polarity markers, including E-Cadherin and Armadillo, apically where they are normally situated. The endogenous Wingless (blue channel) at the D/V boundary is indicated by yellow arrows. Both E-Cadherin-GFP expressed ubiquitously (A, E-CadGFP, red channel) and endogenous Armadillo (B, N27, red channel) are stable in the AJ in spite of very high levels of overexpressed Wingless. The A/P boundary is clearly demarcated by aligned cells (A, white, red or blue arrowheads), and the cells seem more densely packed or apically constricted within the overexpression domain (yellow dimension bars). (C, D) At 10%, Wingless signalling induces accumulation of Armadillo puncta subapically, corresponding to a change in E-cadherin levels. The endogenous Wingless (blue) channel at the D/V boundary is indicated by yellow arrows. (C) and (D) represent the same wing disc at the same basal position to allow comparison of protein localisation. (C) E-Cadherin accumulates in puncta (red circles) but also appears to be depleted from the basolateral membranes (yellow arrows). (D) In contrast, Armadillo accumulates in many puncta, of which many correspond to E-Cadherin dots, but does not appear depleted from the basolateral membrane (compare expression in domain delineated by the yellow dimension bars in A and B).

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Over Expression, Comparison, Membrane, Expressing

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: (A) Similarly to Wingless ligand-dependent signalling, Arm S10 does not affect the distribution of E-Cadherin-GFP (E-CadGFP) in the AJ. (B) In contrast, Arm S10 entirely displaces endogenous Armadillo from the AJ, which can often be identified as puncta immediately subapically (red channel, inset). (C) E-Cadherin-GFP (E-CadGFP) accumulates in puncta upon Arm S10 overpexression similarly to ligand-dependent signalling. (D) In contrast, Arm S10 causes an accumulation of endogenous Armadillo to high levels in the centre of the dppGAL4 overexpression domain, but results in complete loss from the cytoplasm and basolateral membrane at the edges (red arrows).

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Over Expression, Membrane

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: (A) Overexpression of the ΔNArm 1–155 construct with dppGAL4 causes a reduction in E-Cadherin levels concomitant with a decrease in endogenous Armadillo (blue arrows). (B) No change is evident in E-Cadherin levels when the membrane tethered Myr-ΔNArm 1–155 form is overexpressed. (C) Although levels of endogenous Armadillo increase at the level of the AJ upon overexpression of ArmΔC XM19 (red arrows), there is no change in levels of E-Cadherin relative to the wild type (blue arrowheads).

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Over Expression, Construct, Membrane

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: (A) E-Cadherin levels appear reduced in the domain of expression where endogenous Armadillo is excluded. Fewer puncta are also apparent relative to neighbouring wild type tissue upon overexpression of the ΔNArm 1–155 . (B) No change is evident in E-Cadherin levels when the membrane tethered Myr-ΔNArm 1–155 form is overexpressed. (C) E-Cadherin levels are reduced in the domain of ArmΔC XM19 expression, and puncta are lacking. This is accompanied by an increase in the number of N27-postive puncta (red circles), representing endogenous or C-terminally truncated forms of Armadillo. There is also less E-cadherin associated with the anterior stripe, where endogenous Armadillo and E-cadherin perfectly colocalise under wild type conditions (red arrows). Note that in the wing disc in (C) only the dorsal aspect is shown, while (A) and (B) are show the intersection of the A/P and D/V boundaries.

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Expressing, Over Expression, Membrane

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: Levels of endogenous Armadillo (blue), overexpressed mutant (pink) and E-Cadherin (green) were assessed in (adherens) junctional, “cellular”, basolateral (membrane) and nuclear compartments. The “cellular” compartment assesses levels across a field of cells encompassing the basolateral membrane, nucleus and cytoplasm. Mutant constructs that were assessed are illustrated to the left of the graphs, and included Arm S10 , ΔNArm 1–128 , ΔNArm 1–155 , Myr-ΔNArm 1–155 and ArmΔC XM19 . +1 and −1 represent maximal proportion changes in levels above and below the wild type baseline value of 0. Empty boxes indicate no change from wild type. dppGAL4 driver levels are lower in “lateral” relative to “central” domains of expression, as reflected by spotted versus solid colours. E-Cadherin levels were not assessed in the nuclear compartment (N/A).

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Mutagenesis, Membrane, Construct, Expressing

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: The extent of the overexpression domain is indicated with dimension lines, as assessed by staining (not shown here). (A) Although F-Actin staining clearly indicates the aligned cells at the A/P boundary with Arm S10 , no change in levels was observed. (B) In contrast, overexpression of ΔNArm 1–155 results in stretching of the A/P cells and an increase of F-Actin (red arrowhead), corresponding to a region of low E-Cadherin expression (blue arrowhead). Note the folding of the epithelium which reveals peripodial membrane cells. (C) Similarly, Myr-ΔNArm 1–155 causes stretching and F-Actin accumulation at the A/P boundary where E-Cadherin is lower (red and blue arrowheads), and even appears to induce boundary cell-like behaviour at the anterior extent of mutant overexpression (yellow asterisk). (D) Basally, Myr-ΔNArm 1–155 uniquely causes the formation of filopodia, the base of which are associated with F-Actin bundles (inset).

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Over Expression, Staining, Expressing, Membrane, Mutagenesis

Journal: PLoS ONE

Article Title: Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

doi: 10.1371/journal.pone.0002893

Figure Lengend Snippet: In a wild type cell in its basal state (no signalling, left), Armadillo (blue dots) and E-Cadherin (green dots) are targeted to the membrane, possibly via the exocyst complex . From there, Armadillo/E-Cadherin cycle to the Adherens Junction (AJ, box), or are hypothesised to remain in the cytoplasm as a complex free from degradation. There is little Armadillo in the nucleus (grey oval). During Wingless signalling (right), Armadillo accumulates in subapical puncta and can enter the nucleus (blue oval) to activate Wg targets. We hypothesise that Armadillo/E-Cadherin are released from the AJ (black arrows); alternatively the rate of cycling of Armadillo/E-Cadherin may also be increased. In our proposed model, either in its basal state or during signalling, Armadillo/E-Cadherin may be able to bind α-catenin (yellow hexagon). The activated N-terminal deletion mutants (centre, red boxes and dots) represent a range of effects in the spectrum between the basal state and Wingless signalling, depending at least in part on overexpression levels (bottom, red bar). ΔNArm 1–128 (centre, top left) most closely resembles the basal state, with little Armadillo in the nucleus or signalling. At highest levels of overexpression, ΔNArm 1–128 excludes Armadillo from the basolateral membrane and AJ, and is unlikely to efficiently bind α-catenin (grey hexagon; ). Arm S10 (centre, top right) excludes Armadillo from the AJ, as well as basolateral membranes at lowest expression levels. However, Arm S10 has no effect on E-Cadherin levels and should have the capacity to bind α-catenin , , thus ensuring appropriate adhesive function, as well as signalling (red and blue nucleus). In this case, we propose that Armadillo/E-Cadherin at the basolateral membrane may not be sufficiently stable to enter the AJ (grey arrow). Several sources indicate that Armadillo/E-Cadherin is first targeted to the basolateral membrane, and from there to the AJ , . Myr-ΔNArm 1–155 , while unable to bind α-catenin , nevertheless ensures adhesive function through Armadillo/E-Cadherin at the AJ. These would be able to efficiently cycle between the subcellular compartments and enter the nucleus (blue oval). Additionally, filopodia extend into the environment from the basal side of cells expressing Myr-ΔNArm 1–155 . ΔNArm 1–155 , in contrast, impedes proper Armadillo/E-Cadherin function at the basolateral membrane and AJ through a reduction in their levels (fewer blue dots, pale green). It is unable to bind α-catenin, but enters the nucleus where it may interact with the transcriptional machinery. Note that all representations of cytoplasmic protein localisation are inferred from changes in the other more easily quantifiable compartments (e.g. “cellular” = nucleus+basolateral membrane+cytoplasm).

Article Snippet: Primary antibodies used in this study included α-Armadillo “Arm” (Rabbit; 1∶1000; a gift from H. Müller); α-Armadillo “N27” (N27A1; Mouse; 1∶30; Developmental Studies Hybridoma Bank); α-E-Cadherin “Cad2” (Rat; 1∶20; DSHB); α-Scribble “Scrib” (Rabbit; 1∶2000; a gift from N. Gorfinkiel); α-FasciclinIII “FasIII” (Mouse; 1∶50; DSHB); α-Wingless “4D4” (Mouse; 1∶200; DSHB) and α-Wingless “Wg” (Rabbit; 1∶200; a gift from S. Cumberledge); α-HA “Flu”(Flu); α-Neurexin IV (Rabbit; 1∶1000; a gift from N. Gorfinkiel).

Techniques: Membrane, Over Expression, Expressing, Adhesive

Journal: Journal of proteomics

Article Title: Development of an AlphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker.

doi: 10.1016/j.jprot.2012.05.015

Figure Lengend Snippet: Fig. 4 – Screening of fucosylated glycoproteins in the pooled serum samples from normal controls (N) and serum samples from 20 individual patients. Serum levels of total or fucosylated haptoglobin (A), alpha-1 antitrypsin (B), E-cadherin (C), and complement C3c (D) were analyzed by Western blotting and AAL staining, respectively.

Article Snippet: Western blots After blocking for 60 min at room temperature with 3% skimmed milk in TBS (26 mM Tris–HCl, 150 mM NaCl, pH 7.5), the membrane was incubated overnight at 4 °C with goat antibodies against haptoglobin (Abcam) or against α-1-antitrypsin, complement C3c, or E-cadherin (all from Santa Cruz) at a 1:2000 dilution in antibody dilution buffer [1% skimmed milk in TBS containing 0.1% Tween 20 (TBST), pH 7.5], then for 60 min at room temperature with anti-goat IgG antibody (Santa Cruz, 1:2000 dilution in antibody dilution solution) and bound antibodies were detected using enhanced chemiluminescent detection reagent (Millipore) and autoradiography. ii.

Techniques: Western Blot, Staining

Journal: Journal of proteomics

Article Title: Development of an AlphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker.

doi: 10.1016/j.jprot.2012.05.015

Figure Lengend Snippet: Fig. 5 – Survival rates of groups of lung adenocarcinoma patients with a fucosylated index (FI) of E-cadherin>1.5 or ≤1.5 compared by the Kaplan-Meier method. (A) All 154 patients, (B) 35 Early stage (IA, IB, and II) patients, (C)119 Late stage (IIIA, IIIB, and IV) patients.

Article Snippet: Western blots After blocking for 60 min at room temperature with 3% skimmed milk in TBS (26 mM Tris–HCl, 150 mM NaCl, pH 7.5), the membrane was incubated overnight at 4 °C with goat antibodies against haptoglobin (Abcam) or against α-1-antitrypsin, complement C3c, or E-cadherin (all from Santa Cruz) at a 1:2000 dilution in antibody dilution buffer [1% skimmed milk in TBS containing 0.1% Tween 20 (TBST), pH 7.5], then for 60 min at room temperature with anti-goat IgG antibody (Santa Cruz, 1:2000 dilution in antibody dilution solution) and bound antibodies were detected using enhanced chemiluminescent detection reagent (Millipore) and autoradiography. ii.

Techniques:

Journal: Journal of proteomics

Article Title: Development of an AlphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker.

doi: 10.1016/j.jprot.2012.05.015

Figure Lengend Snippet: Fig. 6 – AlphaLISA assay. (A) Effect of biotin-AAL concentration on the AlphaLISA signal for the detection of fucosylated E-cadherin. Different volumes of serum were preincubated with different concentrations of biotinylated-AAL and anti-E-cadherin antibody-protein A acceptor beads for 60 min at room temperature before addition of streptavidin donor beads and incubation for a further 60 min at room temperature before reading the plate. (B) Effect of biotin–anti-E-cadherin monoclonal antibody concentration on the AlphaLISA signal for detection of total E-cadherin. Different volumes of serum were preincubated with different concentrations of biotin-anti-E-cadherin monoclonal antibody and rabbit anti-E-cadherin polyclonal antibody-anti-rabbit IgG-conjugated acceptor beads for 60 min at room temperature before addition of streptavidin donor beads and incubation for a further 60 min at room temperature before reading the plate. (C) The concentrations of 1 μM biotin-AAL and 10 μM biotin-α-E-cadherin monoclonal antibody were chosen for FI evaluation using a 1:50 serum dilution. The fucosylated index of E-cadherin in serum samples from 30 patients with early or late stage lung adenocarcinoma and controls measured by AlphaLISA.

Article Snippet: Western blots After blocking for 60 min at room temperature with 3% skimmed milk in TBS (26 mM Tris–HCl, 150 mM NaCl, pH 7.5), the membrane was incubated overnight at 4 °C with goat antibodies against haptoglobin (Abcam) or against α-1-antitrypsin, complement C3c, or E-cadherin (all from Santa Cruz) at a 1:2000 dilution in antibody dilution buffer [1% skimmed milk in TBS containing 0.1% Tween 20 (TBST), pH 7.5], then for 60 min at room temperature with anti-goat IgG antibody (Santa Cruz, 1:2000 dilution in antibody dilution solution) and bound antibodies were detected using enhanced chemiluminescent detection reagent (Millipore) and autoradiography. ii.

Techniques: Concentration Assay, Incubation

Journal: Journal of proteomics

Article Title: Development of an AlphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker.

doi: 10.1016/j.jprot.2012.05.015

Figure Lengend Snippet: Fig. 7 – Comparison of the FI for E-cadherin in lung cancer sera using the AlphaLISA method or the Western blot and lectin staining method.

Article Snippet: Western blots After blocking for 60 min at room temperature with 3% skimmed milk in TBS (26 mM Tris–HCl, 150 mM NaCl, pH 7.5), the membrane was incubated overnight at 4 °C with goat antibodies against haptoglobin (Abcam) or against α-1-antitrypsin, complement C3c, or E-cadherin (all from Santa Cruz) at a 1:2000 dilution in antibody dilution buffer [1% skimmed milk in TBS containing 0.1% Tween 20 (TBST), pH 7.5], then for 60 min at room temperature with anti-goat IgG antibody (Santa Cruz, 1:2000 dilution in antibody dilution solution) and bound antibodies were detected using enhanced chemiluminescent detection reagent (Millipore) and autoradiography. ii.

Techniques: Comparison, Western Blot, Staining

Journal: Journal of proteomics

Article Title: Development of an AlphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker.

doi: 10.1016/j.jprot.2012.05.015

Figure Lengend Snippet: Fig. 8 – Molecular functions related to non-small cell lung cancer associated with E-cadherin. The web diagram generated using Ingenuity Pathway Analysis software shows the biological functions (Fx) of E-cadherin associated with non-small cell lung cancer.

Article Snippet: Western blots After blocking for 60 min at room temperature with 3% skimmed milk in TBS (26 mM Tris–HCl, 150 mM NaCl, pH 7.5), the membrane was incubated overnight at 4 °C with goat antibodies against haptoglobin (Abcam) or against α-1-antitrypsin, complement C3c, or E-cadherin (all from Santa Cruz) at a 1:2000 dilution in antibody dilution buffer [1% skimmed milk in TBS containing 0.1% Tween 20 (TBST), pH 7.5], then for 60 min at room temperature with anti-goat IgG antibody (Santa Cruz, 1:2000 dilution in antibody dilution solution) and bound antibodies were detected using enhanced chemiluminescent detection reagent (Millipore) and autoradiography. ii.

Techniques: Generated, Software

Journal: Bioengineered

Article Title: MicroRNA MiR-490-5p suppresses pancreatic cancer through regulating epithelial-mesenchymal transition via targeting MAGI2 antisense RNA 3

doi: 10.1080/21655979.2021.2024653

Figure Lengend Snippet: MiR-490-5p targeting MAGI2-AS3 to regulate epithelial–mesenchymal transition (EMT) process. (a) Overexpression of miR-490-5p in cells caused by transfection of miR-490-5p mimic. (b) Up-regulation of miR-490-5p down-regulated the expression of MAGI2-AS3, which was detected by qRT-PCR. (c-d) The mimic group had the lowest protein levels of Matrix metalloproteinase (MMP)2, MMP9 and N-cadherin, and the highest protein level of E-cadherin, while the opposite condition appeared in MAGI2-AS3 group, which were measured by Western blot. ** p < 0.001, vs. control; ^^ p < 0.001 vs. NC; ## P < 0.001, vs. MAGI2-AS3; ΔΔ p <0.001, vs MAGI2-AS3+ mimic.

Article Snippet: Then, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane which was blocked for 1 h with 5% nonfat milk and incubated with primary antibodies against Matrix metalloproteinase (MMP)2 (1:1000, 74kD, ab215986, Abcam), MMP9 (1:1000, 78kD, ab219372, Abcam), E-cadherin (1:10,000, 97kD, ab40772, Abcam), N-cadherin (1:1000, 130kD, ab18203, Abcam) and GAPDH (1:10,000, 36kD, ab181602, Abcam) at 4°C overnight, followed by the incubation with secondary antibody goat anti-rabbit IgG H&L (1:1000, ab205718, Abcam) for 1 h. Then, the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., MD, USA) was used to analyze proteins expressions.

Techniques: Over Expression, Transfection, Expressing, Quantitative RT-PCR, Western Blot