Journal: Oncotarget

Article Title: NFkB is essential for activin-induced colorectal cancer migration via upregulation of PI3K-MDM2 pathway

doi: 10.18632/oncotarget.16343

Figure Lengend Snippet: (A) A spectrum of colon cancer cells lines including HCT116 lacking the ACVR2 receptor, HCT116+chr2 supplemented with the ACVR2 receptor, ACVR2 positive FET cells and SMAD4 null ACVR2 positive SW480 cell lines were treated as follows: Light gray bars=vehicle control; White bars = 25 ng/ml activin for 6 hours; Dark Gray bars = combination treated cells were pre-incubated with wtNBD peptide (10uM) for 45 min and then stimulated with 25ng/ml activin for 6h under serum free condition and Black Bars = treatment of wtNBD peptide (10uM) alone. The cells were analyzed by transwell migration assay and data are expressed as number of particles per high powered field (hpf) as described in Material and Methods. Results are mean SD of three different experiments. (B) Inhibition of NFkB reduces activin-induced upregulation of EMT markers in FET colon cancer cells. The cells were pretreated with wtNBD peptide (10uM) for 45min prior to activin stimulation (25ng/ml) for 72h under serum free condition. Activin treatment induces an increase in expression of mesenchymal markers, vimentin and Snail followed by a decrease in epithelial marker E-Cadherin as examined by western blotting. The above effects were reversed following NFkB inhibition under similar context of activin treatment. The experiments were repeated three times and similar results were obtained. GAPDH was used as a loading control. (C) HCT116 and HCT116+chr2, FET and SW480 colon cancer cell lines were treated as indicated above. Relative viability was assessed by MTT assay as described in Materials and Methods. Data were normalized to control. Results are mean SD of three independent experiments. *p indicates versus control; #p indicates versus activin. */#p < 0.05; **/##p < 0.01 respectively.

Article Snippet: Mitochondrial activity was measured with the 3-(4, 5-dimethythiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay (cell counting kit-8, Dojindo Molecular Technologies, MD, USA) as previously described [ ].

Techniques: Control, Incubation, Transwell Migration Assay, Inhibition, Expressing, Marker, Western Blot, MTT Assay

Journal: The Journal of Biological Chemistry

Article Title: Glycogen Synthase Kinase 3? Interaction Protein Functions as an A-kinase Anchoring Protein *

doi: 10.1074/jbc.M109.047944

Figure Lengend Snippet: GSKIP forms a ternary complex with regulatory RIIα subunits of PKA and GSK3β and facilitates PKA phosphorylation of GSK3β. A, left panel, wells of microtiter plates were coated with His-GSKIP (500 nm) and incubated with GSK3β (0.25–64 nm) in blocking buffer or with blocking buffer without GSK3β. GSK3β was detected with anti-GSK3β and secondary HRP-conjugated antibodies and an HRP-catalyzed reaction with a chromogenic substrate solution. Right panel, wells of microtiter plates were coated with RIIα (50 nm) in blocking buffer or with blocking buffer in the absence of RIIα. GSKIP (250 nm) in combination with increasing amounts of GSK3β (0.25–64 nm) was added. To quantify unspecific signals, the GSKIP-GSK3β-combination was incubated in wells blocked in the absence of RIIα (▵). Direct binding of GSK3β to RIIα was assessed by incubation of RIIα-coated wells with GSK3β (0.25–64 nm; ○). Detection was carried out as above. B, left panel, CFP or CFP-GSKIP was transiently expressed in HEK293 cells. The cells were left untreated, incubated with the direct activator of adenylyl cyclases, forskolin (20 μm, 30 min), or pretreated with the PKA inhibitor H89 (30 μm, 30 min prior to FSK treatment). Cells were lysed, and CFP-GSKIP, phosphorylated (p-GSK3β), and total GSK3β were detected with specific antibodies by Western blotting (WB). Right panel, signals obtained for p-GSK3β and GSK3β by Western blotting were analyzed densitometrically. The ratio of p-GSK3β to GSK3β was calculated (n = >5 independent experiments for each experimental condition, mean ± S.E.). C, left panel, HEK293 cells were treated with FSK or a combination of FSK and H89 as in B or with stearate-coupled GSKIPtide, which displaces GSK3β from GSKIP (20), or the inactive control peptide GSKIPtide-L130P (100 μm, 30 min). Cells were lysed and phosphorylated GSK3β (p-GSK3β) and GAPDH (loading control) were detected by Western blotting. Right panel, signals obtained for p-GSK3β and GAPDH by Western blotting were analyzed densitometrically. The ratio of p-GSK3β to GAPDH was calculated (n = 7 independent experiments for each experimental condition, mean ± S.E.). B and C, significantly different from untreated control: #, p < 0.05; ##, p < 0.01; ###, p < 0.001. Significant differences between groups connected by lines: *, p < 0.05; **, p < 0.01). n.s., not significant.

Article Snippet: Specific antibodies were isolated by affinity chromatography of the antisera using GSKIP immobilized on thiopropyl-Sepharose 6B (GE Healthcare) ( 24 ).

Techniques: Phospho-proteomics, Incubation, Blocking Assay, Binding Assay, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Glycogen Synthase Kinase 3? Interaction Protein Functions as an A-kinase Anchoring Protein *

doi: 10.1074/jbc.M109.047944

Figure Lengend Snippet: GSKIP functions as an AKAP in vitro. A, GSKIP and the DD domain of human regulatory RIIα subunits of PKA were generated and NMR measurements performed. 1H-15N heteronuclear single quantum coherence spectra of the GSKIP domain in the absence (red) or presence of DD (black) at 1:0.5 ratio are shown. Disappearance of the GSKIP resonances is caused because of chemical exchange broadening and/or increased transverse relaxation rate due to high molecular weight of the complex. Lower panel, Protein Data Bank structure of GSKIP (Protein Data Bank code 1sgo) The RII-binding domain (black) encompasses the amphipathic α1-helix of GSKIP. The conserved hydrophobic amino acid residues Ala-37, Val-40, Val-41, Val-44, Leu-45, and Val-48 (see also Fig. 1A) are depicted as ball-and-stick models. The contact interface for RII binding is hidden by a β-sheet. B, surface plasmon resonance measurements to determine the association and dissociation rate constants (see Table 2) for the binding of GSKIP to PKA regulatory RII subunits. Human RIIα and RIIβ subunits (upper and lower panel, respectively) were captured on 8-AHA-cAMP sensor chips, and His-GSKIP was injected into the Biacore instrument in the indicated concentrations. The plots show representative experiments. Each experiment was repeated at least three times using different protein preparations and different immobilization rates of the regulatory subunits. RU, resonance units. C, immunoprecipitated (IP) GSKIP binds RII subunits. Cyan fluorescent protein (CFP; lane 1) and the indicated fusions of human full-length GSKIP with CFP (lane 2) and GSKIP-V41P/L45P with CFP (lane 4) were transiently expressed in HEK293 cells. CFP-GSKIP-V41P/L45P contains prolines in the RII-binding domain disrupting the α-helical structure and thus preventing the interaction with RII. The cells were lysed, and GSKIP was immunoprecipitated with anti-GSKIP antibodies, or the precipitation was carried out with the corresponding preimmune serum. Precipitated proteins were probed for RII binding by RII overlay assay in the absence (control) or in the presence of the PKA anchoring disruptor peptide, AKAP18δ-L314E (10 μm). Signals were detected by autoradiography. Lane 3, molecular weight standard. Lower panel, Western blot (WB) with anti-green fluorescent protein antibody (also detects CFP (26)) for the detection of CFP, CFP-GSKIP, or CFP-GSKIP-V41P/L45P in HEK293 cells transiently expressing the proteins. Proteins were detected in precipitates obtained with rabbit anti-GSKIP antibody (IP, left panel) or obtained with preimmune serum (middle panel) or in cell lysates.

Article Snippet: Specific antibodies were isolated by affinity chromatography of the antisera using GSKIP immobilized on thiopropyl-Sepharose 6B (GE Healthcare) ( 24 ).

Techniques: In Vitro, Generated, High Molecular Weight, Binding Assay, SPR Assay, Injection, Immunoprecipitation, Overlay Assay, Control, Autoradiography, Molecular Weight, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Glycogen Synthase Kinase 3? Interaction Protein Functions as an A-kinase Anchoring Protein *

doi: 10.1074/jbc.M109.047944

Figure Lengend Snippet: GSKIP is widely expressed and functions as an AKAP in vivo. A, lysates from the indicated rat organs were subjected to Western blot (WB) analysis with an anti-GSKIP antibody and, as a loading control, anti-GAPDH antibody (167 μg of total protein per lane). B, cAMP-agarose pulldowns were obtained from the indicated rat tissue lysates (3 mg of total protein in each sample) in the absence (− cAMP) or presence (+ cAMP; 50 μm) of cAMP. RIIα subunits of PKA and GSKIP were detected with specific antibodies by Western blotting. skel., skeletal. C, indicated subcellular fractions were obtained from SH-SY5Y cells. 10 μg of total protein from each fraction was analyzed for the presence of the indicated proteins by Western blotting. D, GSKIP and RIIα co-localize in HEK293 cells. HEK293 cells were transiently transfected to express YFP-RIIα and fusions of CFP with wild type GSKIP (CFP-GSKIP WT), GSKIP-V41P/L45P, or as a plasma membrane marker AKAP18α.

Article Snippet: Specific antibodies were isolated by affinity chromatography of the antisera using GSKIP immobilized on thiopropyl-Sepharose 6B (GE Healthcare) ( 24 ).

Techniques: In Vivo, Western Blot, Control, Transfection, Clinical Proteomics, Membrane, Marker

Journal: The Journal of Biological Chemistry

Article Title: Glycogen Synthase Kinase 3? Interaction Protein Functions as an A-kinase Anchoring Protein *

doi: 10.1074/jbc.M109.047944

Figure Lengend Snippet: GSKIP contains the evolutionarily conserved DUF727, which binds PKA and GSK3β. A, RII-binding domain of GSKIP is located within DUF727. Shown are sequence alignments of the RII-binding domain of GSKIP with orthologues from the indicated vertebrate and invertebrate species (upper and lower, panels, respectively). Swiss-Prot protein identification codes (Prot.-ID) and positions of the RII-binding domains within the cognate proteins are indicated (for Canis familiaris, the NCBI protein accession is given as no Swiss-Prot entry was available). Amino acid residues in conserved positions of RII-binding domains are shaded in gray (see also Fig. 1A). Peptides depicted in the alignments were spot-synthesized and assayed for RII binding in the absence (control) or the presence of the PKA anchoring disruptor peptide AKAP18δ-L314E (10 μm). Signals were detected by autoradiography. Numbers of the spots correspond to the respective numbers in the alignments. Shown are representative membranes from three independent experiments. B, GSK3β binding is conserved in GSKIP orthologues. Left panel, alignment of all 51 DUF727 proteins with the C terminus of human GSKIP (amino acids 115–139) and human axin-1 and -2. Hydrophobic residues required for GSK3β interaction, which are identical (dark gray) or similar (bright gray) to human axin-1, are highlighted. Right panel, peptide spots of corresponding sequences in the alignment were spot-synthesized and incubated with GST-GSK3β or GST as a negative control. Peptides marked with a P contain an Leu → Pro (L→P) mutation in the position corresponding to amino acid 130 in human GSKIP (asterisk in left panel). Protein binding was detected with an anti-GST antibody. Both axin peptides and 40 of 50 DUF727 peptides bound GST-GSK3β. This result is representative for three independent experiments.

Article Snippet: Specific antibodies were isolated by affinity chromatography of the antisera using GSKIP immobilized on thiopropyl-Sepharose 6B (GE Healthcare) ( 24 ).

Techniques: Binding Assay, Sequencing, Synthesized, Control, Autoradiography, Incubation, Negative Control, Mutagenesis, Protein Binding

Journal: Parasites & Vectors

Article Title: A mitochondrial HSP70 (HSPA9B) is linked to miltefosine resistance and stress response in Leishmania donovani

doi: 10.1186/s13071-016-1904-8

Figure Lengend Snippet: Effect of HSPA9B overexpression on differentiation. a L. donovani WT and transgenic promastigotes (HSPA9B-mCherry) were inoculated in low pH (pH = 5.5) medium and 37 °C to trigger differentiation to axenic amastigotes. Cells were collected 24 and 48 h post-induction of differentiation and either lysed for western blotting with axenic amastigote A2 marker and anti-tubulin as a loading control or counted using a Neubauer chamber. b The molecular weight of standard proteins is indicated in kDa and the mean ± standard deviation (SD) of triplicate determinations are shown, respectively

Article Snippet: Crude cell lysates, digitonin enriched and membrane and lumen organelle enriched fractions were separated in 4–12% Bis-Tris NuPAGE gels (Life, Carlsbad, USA) and revealed using the following antibodies: anti-GFP-horseradish-peroxidase (HRP)-conjugated antibody (Miltenyi, Bergisch Gladbach, Germany), rabbit polyclonal anti-mCherry antibody (Abcam, Cambridge, UK), mouse monoclonal anti-A2 antibody (Abcam, Cambridge, UK), mouse monoclonal anti-tubulin antibody (Sigma-Aldrich, St. Louis, USA), rabbit polyclonal anti-lipoic acid antibody (Abcam, Cambridge, UK) and anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (Pierce, Waltham, USA).

Techniques: Over Expression, Transgenic Assay, Western Blot, Marker, Molecular Weight, Standard Deviation

Journal: Nature Communications

Article Title: Reduced secretion of neuronal growth regulator 1 contributes to impaired adipose-neuronal crosstalk in obesity

doi: 10.1038/s41467-022-34846-w

Figure Lengend Snippet: a Experimental workflow showing anatomical localisation of AT depots and subsequent procedures performed on AT explants. b Upper panel: H&E stainings of tissue sections. Lower panel: immunofluorescence images of AT sections stained for perilipin and UCP1. Nuclei are counterstained with DAPI. Scale bars: 50 µm. Insets show higher magnifications of randomly selected areas. Representative pictures of 1 out of 2 replicates. c Western blot of representative AT lysates (2 out of 4 replicates) stained for perilipin and UCP1. A brain lysate was loaded as negative control for AT-specific proteins. d Western blot of representative AT lysates (1 out of 2 replicates) and corresponding supernatants stained for β-actin, adiponectin, perilipin and UCP1. e , f Unsupervised hierarchical clustering (heatmap, e ) and principal component analysis ( f ) of the 363 consistently identified secreted proteins detected by MS in the conditioned media of different AT depots. Hierarchical clustering was performed using the complete agglomeration method, Z -score-scaled abundances are displayed. N = 6 biological replicates per group. AT adipose tissue, AR PVAT aortic arch perivascular AT, TH PVAT thoracic PVAT, AB PVAT abdominal PVAT, VI WAT visceral white AT, SC WAT subcutaneous white AT, BAT interscapular brown AT, UCP1 uncoupling protein 1, M molecular weight marker.

Article Snippet: The following primary antibodies were used for immunostainings: Perilipin-1 ab61682, abcam, 1:200; UCP1 ab23841, abcam, 1:100; Ncam1 ab220360, abcam, 1:100; Chl1, AF2147, R&D, 1:100; L1cam ab208155, abcam, 1:100; tyrosine hydroxylase AB152, Merck, 1:100; beta3-adrenergic receptor ab94506, abcam, 1:100; Negr1 AF5384, R&D, 1 μg/ml.

Techniques: Immunofluorescence, Staining, Western Blot, Negative Control, Molecular Weight, Marker

Journal: Developmental biology

Article Title: Role of presenilin-1 in cortical lamination and survival of Cajal-Retzius neurons.

doi: 10.1016/j.ydbio.2004.09.024

Figure Lengend Snippet: Fig. 4. Expression of glial markers, RC2, BLBP, and Nestin, is normal at E12.5 and progressively diminishes in PS1 cKO mice. (A–L) Confocal images captured from the rostral medial telencephalon of embryonic brains immunostained for RC2 (A–J) or BLBP (K, L) are shown. (A–D) At E11.5 and E12.5, immunohistochemical analysis shows similar RC2 immunoreactivity in the comparable area of the telencephalon in PS1 cKO and control mice, indicating unaffected radial glia at this age. (E, F) At E13.5, RC2 immunoreactivity is reduced in PS1 cKO brains, indicating reduction in radial glia. Comparable areas of the PS1 cKO and control telencephalon are shown. (G, H) At E15.5, RC2 levels appear further diminished in the PS1 cKO telencephalon, with shorter glial processes extending toward the pial membrane and more intense RC2 staining near the lateral ventricle. (I–L) At E17.5, RC2 immunoreactivity is markedly reduced in the telencephalon of PS1 cKO mice. The decrease in radial glia is confirmed using another marker for radial glia, BLBP, the expression of which is upregulated upon contact with migrating neurons. Comparable areas of the PS1 cKO and control telencephalon are shown. (M, N) BLBP protein levels are relatively normal in PS1 cKO brain at E12.5, but decrease to 82% of control levels by E17.5 (P = 0.007). Nestin levels are slightly reduced in mutants versus controls at E12.5 (92%; P = 0.019) and diminish to 54% of control levels at E17.5 (P = 0.001). Alpha-spectrin, a 240-kDa structural protein, was used as a control for Nestin Western blots, since its molecular weight is similar to Nestin (approximately 200 kDa). Error bars represent standard deviation. The P values are indicated as follows: P b 0.05 (one asterisk); P b 0.01 (two asterisks) as evaluated by Student t test. Scale bar: 100 Am.

Article Snippet: For detection of PS1 and BLBP, lysates were loaded onto either 4–20% (PS1) or 12% (BLBP) Tris–glycine SDS–PAGE gels and transferred to PVDF membranes (BioRad, Hercules, CA).

Techniques: Expressing, Immunohistochemical staining, Control, Membrane, Staining, Marker, Western Blot, Molecular Weight, Standard Deviation

Journal: Poultry Science

Article Title: Efficacy of a plant-produced infectious bronchitis virus-like particle vaccine in specific pathogen-free chickens

doi: 10.1016/j.psj.2023.102953

Figure Lengend Snippet: SDS-PAGE (A) and Western blot (B) of plant-produced spike protein (synthetic construct mIBV-S2P-NDV-F TM/CT ) coinfiltrated at a 4:1 ratio with the NDV matrix protein. Lane 1: molecular weight marker; Lane 2: plant-expressed empty pEAQ-HT vector; Lane 3: purified live QX-like IBV strain ck/ZA/3665/11; Lanes 4 to 7: Fractions 3 to 6 extracted in PBS buffer; Lanes 8 to 11: Fractions 3 to 6 extracted in Bicine buffer; Lanes 12 to 15: Fractions 3 to 6 extracted in Tris buffer.

Article Snippet: The fractions containing the highest level of S protein expression as confirmed by SDS-PAGE and Western blot were pooled (i.e., those for mIBV-S2P-NDV-F TM/CT :Matrix processed in PBS buffer), dialyzed in 1× PBS using 3500 mW CO Slide-A-Lyzer Dialysis Cassettes (ThermoFisher Scientific, Waltham, MA), and stabilized with 15% (w/v) trehalose dehydrate (Sigma-Aldrich).

Techniques: SDS Page, Western Blot, Produced, Construct, Molecular Weight, Marker, Plasmid Preparation, Purification

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 1. Nucleolar translocation of RelA and proteasome activity. A, SW480 colon cancer cells were treated with aspirin (0–5 mmol/L, 16 h). 20S proteasome activity was measured in whole-cell lysates using fluorometric assay. Columns, percentage of basal (0 mmol/L) activity (n = 4); bars, SE. B, SW480 cells were treated with 10 mmol/L aspirin for the times specified and then 20S proteasome activity was measured as above. Columns, percentage of basal (0 h) activity (n = 4); bars, SE. C, SW480 cells were transfected with HA-tagged ubiquitin 24 h before treatment with aspirin (10 mmol/L) for the specified times. Anti-HA Western blot analysis was done on whole-cell extracts. Actin is used as a protein loading control. D, SW480 cells treated with aspirin (0 or 5 mmol/L, 16 h) were fractionated using sucrose gradients. 20S proteasome activity was measured in lysates from the nuclear and nucleolar fractions as above. Columns, percentage of nontreated (NT) nuclear proteasome activity (n = 3); bars, SE. Inset, anti-C23, fibrillarin (nucleolar markers), and lamin B (nonnucleolar protein) Western blot analysis done on lysates from the nuclear and nucleolar fractions.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Translocation Assay, Activity Assay, Transfection, Ubiquitin Proteomics, Western Blot, Control

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 2. Ubiquitination of RelA precedes nucleolar translocation of the protein. A, SW480 cells were treated with aspirin (0–5 mmol/L, 16 h). Top, the levels of high molecular weight [indicative of ubiquitination (Ub.)] and native RelA were determined by Western blot analysis of whole-cell lysates. Bottom, RelA was immunoprecipitated (IP) from lysates, and recovered protein analyzed using anti-ubiquitin Western blot analysis (WB Ub.). Immunoprecipitation with isotyped IgG controlled for specificity. Stripped gels were reprobed for RelA to monitor the amount of RelA immunoprecipitated (WB. RelA). B, SW480 cells were transfected with WT 6His-ubiquitin (ub) or the R7 mutant that cannot form polyubiquitin chains. Twenty-four hours after transfection, cells were either left untreated (NT) or treated with aspirin (5 mmol/L) or MG132 (25 μmol/L) for a further 16 h. His-tagged proteins were precipitated from whole-cell lysates using Ni-agarose beads and then subjected to anti-RelA and anti-histidine (his) Western blot analysis (WB). NS, nonspecific band. C, SW480 cells were treated with aspirin (10 mmol/L) for the times specified. Top, the levels of native and high molecular weight forms of RelA were determined using Western blot analysis. Bottom, the nuclear distribution of RelA was assessed by immunocytochemical staining (magnification, ×63). D, SW480 cells were exposed to UV-C radiation (40 J/m2) or TNF (10 ng/mL) for the times specified. Top, anti-RelA Western blot analysis done on whole-cell lysates. Bottom, immunomicrographs showing the cellular distribution of RelA (magnification, ×63).

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Ubiquitin Proteomics, Translocation Assay, High Molecular Weight, Western Blot, Immunoprecipitation, Transfection, Mutagenesis, Staining

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 3. Proteasome inhibition induces ubiquitination and nucleolar translocation of RelA. A, SW480 cells were treated with carrier (DMSO), MG132 (25 μmol/L), or lactacystin (Lact.; 35 μmol/L) for 16 h. Immunomicrographs (×63) show the cellular localization of RelA. C23 staining identifies nucleoli. DNA is stained by DAPI. The percentage of cells showing nucleolar RelA (as indicated by colocalization with C23) was determined in at least 200 cells from at least five randomly selected fields of view (n = 3). B and D, SW480 cells were treated with MG132 (25 μmol/L) or lactacystin (50 μmol/L) for the times specified. Top, anti-RelA Western blot analysis showing native and high molecular weight forms of the protein, indicative of the addition of multiple ubiquitin chains (Ub. RelA). Middle, the cellular distribution of RelA was determined by immunocytochemical staining (magnification, ×63). Bottom, for each time point, the percentage of cells showing nucleolar localization of RelA (as depicted by areas devoid of DAPI staining) was determined as in A (n = 3). B, bottom, gray line, SW480 cells were transiently transfected with the NF-κB–dependent 3×κB ConA-luc and the control pCMVβ reporter constructs. Twenty-four hours after transfection, cells were treated with MG132 (25 μmol/L) for the times specified. Results were normalized using β-galactosidase activity and are presented as the percentage of relative luciferase activity compared with basal (carrier treated) controls (n = 2); bars, SD. C, SW480 cells were transfected with GFP-RelA 24 h before treatment with MG132 (25 μmol/L, 0 or 2 h). Immunoprecipitation (IP) of ubiquitinated proteins followed by anti-RelA Western blot analysis (WB) of recovered complexes confirmed an increase in ubiquitinated forms of RelA 2 h after MG132 treatment. Control immunoprecipitations were done with preimmune (IgG) serum. RelA in input samples is shown.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Inhibition, Ubiquitin Proteomics, Translocation Assay, Staining, Western Blot, High Molecular Weight, Transfection, Control, Construct, Activity Assay, Luciferase, Immunoprecipitation

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 4. Amino acids 27–30 are required for ubiquitination and nucleolar translocation of RelA. A, SW480 cells were transfected with the specified plasmids and treated with MG132 (25 μmol/L) or lactacystin (35 μmol/L) for 16 h as above; then the cellular localization of GFP-tagged RelA was determined in live cells using an Axiovert 100 inverted fluorescent microscope (×40; left). Nucleoli are indicated by arrows in phase-contrast images. Right images, fixed cells counterstained with DAPI (DNA stain). Nucleoli are depicted by areas devoid of DAPI staining (magnification, ×63). B, SW480 cells were transfected with the specified GFP plasmids, then either left untreated (Asp. −) or treated with aspirin (10 mol/L), carrier (MG132 −), or MG132 (25 μmol/L) for 2 h. The levels of native and high molecular weight (Ub) RelA and total protein ubiquitination were determined by Western blot analysis. C, SW480 cells were transfected and treated with MG132 (25 μmol/L) or aspirin (10 mmol/L) as above. The levels of ubiquitinated RelA were determined by anti-ubiquitin immunoprecipitation (IP) followed by anti-RelA Western blot analysis (WB). Membranes were stripped and reprobed to show the effects of the agents on total protein ubiquitination. Right, SW480 cells constitutively expressing GFP-RelA WT or Δ27–30 were transfected with 6His-ubiquitin. Cells were then treated for 2 h with aspirin (10 mmol/L), and ubiquitinated proteins precipitated using Ni-agarose beads. Precipitated proteins were subjected to anti-RelA and anti-His Western blot analysis. D, SW480 cells were transfected with the specified vectors and then treated with MG132 (25 μmol/L) or lactacystin (0–75 μmol/L) for 16 h. Annexin V-biotin staining, with a Texas red-streptavidin conjugate, was used to identify apoptotic cells. The percentage of cells expressing GFP-tagged RelA undergoing apoptosis was determined by fluorescent microscopy in at least 250 transfected cells for each sample. Columns, mean of at least three independent experiments; bars, SE.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Ubiquitin Proteomics, Translocation Assay, Transfection, Microscopy, Staining, High Molecular Weight, Western Blot, Immunoprecipitation, Expressing

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 5. COMMD1 modulates the nuclear distribution of RelA. A, immunomicrographs (magnification, ×63) showing aspirin (5 mmol/L, 16 h)-mediated nucleolar translocation of RelA in SW480 cells transfected with GST-COMMD1 or control (GST) vector. NT, nontreated. The percentage of cells in the total cell population showing nucleolar RelA (as indicated by colocalization with the nucleolar marker fibrillarin) was determined in at least 200 cells. Columns, mean (n = 4); bars, SE. Inset, anti-COMMD1 immunoblot showing levels of GST-COMMD1 in transfected SW480 cells. B and C, SW480 cells were transfected with control or COMMD1 siRNA (C1/1 and C1/2) and then treated with aspirin (10 mmol/L) for 0 to 8 h. B, anti-RelA immunoblot showing levels of native and ubiquitinylated RelA in response to aspirin (10 mmol/L, 2 h) treatment. Native RelA controls for protein loading. Numbers indicate the mean relative intensity (RI) of high molecular weight RelA (compared with native RelA; quantified using ImageJ; n = 3). C, immunocytochemical staining determined the nuclear distribution of RelA. The percentage of cells showing nucleolar RelA was calculated as above. Inset, Western blot analysis determined the levels of COMMD1 with (+) and without (−) aspirin (10 mmol/L, 2 h) treatment. Actin was used as a control for protein loading. D, SW480 cells were transfected as indicated, then either untreated or treated with aspirin (5 mmol/L) for 24 h. Apoptotic cells were visualized using fluorescent microscopy and the percentage of apoptotic cells within the total cell population was calculated. Columns, average fold increase in apoptosis in response to aspirin compared with the equivalent nontreated control (n = 3); bars, SE.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Translocation Assay, Transfection, Control, Plasmid Preparation, Marker, Western Blot, High Molecular Weight, Staining, Microscopy

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 6. Aspirin modulates the COMMD1-RelA interaction. A, SW480 cells were treated with 10 mmol/L aspirin for the times indicated and COMMD1 protein levels determined by Western blot analysis. Actin was used as a control for protein loading. B, SW480 cells stably expressing GFP-RelA were transfected with either GST-COMMD1 or an empty vector control (Vec-GST). Twenty-four hours after transfection, cells were treated with 10 mmol/L aspirin for the indicated times, and GST-tagged proteins precipitated from whole-cell lysates using glutathione Sepharose (GSH beads). Anti-RelA Western blot analysis (WB) of precipitated proteins confirmed an interaction between COMMD1 and RelA in response to aspirin treatment. Gels were reprobed with GST to examine the levels of precipitated GST-COMMD1 and GST-control. GFP-RelA and GST-COMMD1 levels in input samples are shown. C, SW480 cells were treated with either aspirin (10 mmol/L) or MG132 (25 μmol/L) for 2 h in the presence or absence of calyculin A (50 nmol/L). Anti-RelA immunoblot shows native and ubiquitinated (Ub) protein in whole-cell extracts. The levels of phosphorylated RelA (S468 and S563) were also determined by Western blot analysis. Filamin was used as a control for protein loading. D, model for nucleolar translocation of RelA and NF-κB–regulated apoptosis in colorectal cancer cells. See Discussion for details.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Western Blot, Control, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Translocation Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 1. Common commercially available ADNP antibodies give rise to non-specific binding. HEK293T, HeLa, SHSY-5Y and a lymphoblastoid control cell line (LCL) were lysed in RIPA buffer and used as protein samples for the assessment of the published ADNP antibodies. Samples were blocked and incubated in 5% blocking-grade non-fat dry milk/TBST with the optimized dilution listed in Table 3. The predicted molecular weight of ADNP is 124 kDa. However, only non-specific signals were detectable. GAPDH was used as a loading control. The datasheet of the tested antibodies indicated that whole or nuclear extracts from HeLa cells should be used as a positive control, which fails to raise a reliable ADNP signal in all tested antibody conditions.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Binding Assay, Control, Incubation, Blocking Assay, Molecular Weight, Positive Control

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 2. Verification of the specificity of an N-terminal ADNP antibody (Aviva Systems) by performing a blocking peptide competition assay. (A) HEK293T, HeLa, SHSY-5Y and a control lymphoblastoid cell line (LCL) were lysed in RIPA buffer and used as protein samples for the assessment of N-terminal antibody of Aviva systems in a 1:1000 dilution. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. The antibody recognizes ADNP specifically at 150 kDa in HEK293T, HeLa and SHSY-5Y cell lines, but a faint signal ranging from 75 to 150 kDa in the control LCL. (B) Western blot analysis of the blocking peptide competition assay. Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced the signal detected at 75- 150 kDa in all tested cell lines. Non-specific binding was detected after use of the immunization peptide presenting as a faint signal below the 37 kDa marker.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Blocking Assay, Competitive Binding Assay, Control, Molecular Weight, Western Blot, Concentration Assay, Binding Assay, Marker

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 3. A polyclonal N-terminal ADNP antibody from Aviva Systems detects ADNP specifically in murine and rat tissues and suggests proteolytic processing of the protein in the human brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats and humans were lysed in RIPA buffer and used as protein samples for the assessment of N-terminal antibody of Aviva systems. (A–C) The predicted molecular weight of ADNP is 124 kDa. The antibody recognizes ADNP in a range of 145 kDa with (E) additional lower mass signal of 85 kDa in all human brain regions. (B–D–F) Western blot analysis of the blocking peptide competition assay. Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced the signal observed at 145 kDa in all tested cell lines. Importantly, the 85 kDa band suggestive for proteolytic cleavage as well as degraded ADNP signal disappeared completely after immunization peptide supplementation. GAPDH was used as a loading control.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Control, Molecular Weight, Western Blot, Blocking Assay, Competitive Binding Assay, Concentration Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 4. Three independent commercially available C-terminal polyclonal ADNP antibodies detect ADNP specifically in different in vitro sample materials and show clear instability of the protein. HEK293T, HeLa, SHSY-5Y and a lymphoblastoid cell line (LCL) were lysed in RIPA buffer and used as protein samples for three different C-terminal ADNP antibodies. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. All the tested antibodies recognized ADNP with a molecular weight of 150 kDa. Samples were blocked and incubated in 5% blocking-grade non-fat dry milk/TBST with the optimized dilution listed in Table 3.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: In Vitro, Control, Molecular Weight, Incubation, Blocking Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 5. Different C-terminal ADNP antibodies detect ADNP in the range of 150 kDa and suggest proteolytic processing of the protein in the brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats, and humans were lysed in RIPA buffer and used as protein samples for the assessment with three C-terminal antibodies with the optimized dilutions listed in Table 3. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A)C) Murine samples indicate detection of ADNP in the range of 150 kDa with bands suggesting proteolytic processing at 50 kDa. (D–F) Rat samples indicate detection of ADNP in the range of 150 kDa with bands indicating proteolytic processing at 82 kDa after incubation with the C-terminal Abcam antibody. (G–I) Human brain samples indicate detection of ADNP at different molecular weights of 124 – 150 kDa in the adult frontal lobe and hippocampus and highlight the antibody differences in detection of ADNP. The three tested antibodies showed strong band signals at lower molecular weights, which could indicate proteolytic cleavage or degradation of the protein.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Control, Molecular Weight, Incubation

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 6. Unambiguous detection of ADNP using homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines. mESCs containing either wild-type, homozygous mutants, or complete Adnp knockout were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal ADNP, 3x-DYKDDDDK, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight with additional lower mass signal of 37—65 kDa in Adnp homozygous and parental control mESCs. (B) Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced all signals observed mESC lines, indicating that the N-terminal antibody does not bind ADNP specifically in mESCs. (C) Detection of wild- type and homozygous Adnp mutants by means of a C-terminal 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 150 kDa in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line using a DYKDDDDK antibody. Truncated ADNP mutants, p.Tyr718* and p.Lys407Valfs*31, were detected at a lower molecular weight of 80 kDa, respectively 48 kDa. (D–F) Wild-type ADNP detection by means of three different C-terminal antibodies in mESC lines. Wild-type ADNP was detected with a strong signal at 150 kDa in the parental control line with a rather decreased signal in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line. Disappearance of the 150 kDa band was observed in the mESC line with complete Adnp homozygosity, indicating a reliable molecular weight of 150 kDa for ADNP.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: CRISPR, Knock-Out, Control, Molecular Weight, Concentration Assay, FLAG-tag

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 7. Unambiguous detection of ADNP using an N-terminal GFPSpark and N-DYKDDDDK (Flag) tag expression vector. (A) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP- GFPSpark and mutated constructs using an anti-GFP antibody. (B) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-GFPSpark and mutated constructs using the N-terminal ADNP antibody (Aviva Systems). (C) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP- DYKDDDDK (Flag) and mutated constructs using an anti-DYKDDDDK antibody. (D) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-DYKDDDDK and mutant constructs using the N-terminal ADNP antibody (Aviva Systems). The observed molecular weight of wild-type ADNP-GFPSpark is 175 kDa (including 25 kDa GFPSpark tag), respectively ADNP-DYKDDDDK 150 kDa, with each of their mutants showing a lower molecular weight as a consequence of the truncating mutations. Detection with antibodies for GFP, DYKDDDDK (Flag), and ADNP gave comparable results. GAPDH was used as a loading control in all experiments.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: FLAG-tag, Expressing, Plasmid Preparation, Western Blot, Construct, Mutagenesis, Molecular Weight, Control

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 8. Western blotting of ADNP in a HCT116 colon cancer cell line, carrying the prevalent heterozygous p.Tyr719* mutation. HCT116 cells containing a wild-type and p.Tyr719* mutant allele were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal antibody, 3x-DYKDDDDK, HA-tag, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control in all experiment. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight an additional signal of 45 kDa, indicating proteolytic cleavage or non-specific binding. (B) Administration of the immunization peptide in a 5 × excess to antibody concentration reduced all signals, indicating that the N-terminal antibody does not bind ADNP specifically in HCT116 cells. (C) Detection of wild-type ADNP by means of the 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 182 kDa in the 3xFlag-V5-loxP- neonGreen/3xHA-loxP-mCherry engineered line using a DYKDDDDK antibody, 32 kDa by tag insertion. (D) Detection of mutant ADNP by means of the HA-epitope tag. A truncated mutant p.Tyr719 ADNP protein was detected in at 105 kDa in the 3xFlag-V5-loxP-neonGreen/3xHA-loxP-mCherry engineered line using a HA-antibody, 25 kDa above its predicted molecular weight by tag insertion. Instability of the truncated protein was observed by a degrading smear. (E–G) Wild-type ADNP detection by means of three different C-terminal antibodies. Non-processed ADNP was detected with a strong signal at 150 kDa in the control line and at a molecular weight of 182 kDa in the genome-edited cell line. In both cases, a degrading smear was observed, indicating instability of the wild-type protein.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Western Blot, Mutagenesis, Control, Molecular Weight, Binding Assay, Concentration Assay, FLAG-tag

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 9. Western blotting of ADNP in human induced pluripotent stem cells (hiPSCs), carrying distinct heterozygous ADNP mutations mediated by CRIPSR/Cas9. (A, B) hiPSCs were lysed in RIPA buffer and analyzed by western blotting with the N-terminal antibody (Aviva Systems) with application of our blocking peptide competition assay. Here, no reliable ADNP signal was detected. The molecular weight of the ADNP mutant lines is expected to decrease to 127 kDa for the Asn832Lysfs*81, respectively to 48 kDa for the lys408Valfs*31 line. However, no signal is observed at the predicted weight for the mutations. (C–E) The C-terminal antibodies of Protein Technology, Abcam, and the Sarma Laboratory were able to visualize wild- type ADNP at 150 kDa. Possessing the desired epitope for mutant ADNP detection, the C-terminal antibody of Protein technology was not able to capture the predicted truncated protein. GAPDH was used as a loading control. (F) The ADNP signal was quantified determining the ratio of the wild-type protein in mutant to control cell lines. Here, the relative ADNP expression decreased in the Asn832Lysfs*81 cell line compared to the control, whereas mutant-to-wild-type expression ratio showed a higher signal with the antibodies of Protein Technology and Abcam in the lys408Valfs*31 cell line.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Western Blot, Blocking Assay, Competitive Binding Assay, Molecular Weight, Mutagenesis, Control, Expressing

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 10. Absence of a mutant ADNP protein after immunoblotting of different lymphoblastoid cell lines from Helsmoortel-Van der Aa syndrome patients. (A) LCLs of four control subjects and six patients were lysed in RIPA buffer and analyzed by western blotting with the N-terminal antibody (Aviva Systems). The expected wild-type ADNP signal presented at 150 kDa together with two non-specific bands at 50 kDa and 75 kDa with no difference in expression (p = 0.42; ns) of the wild-type protein. However, the ADNP mutants at a lower molecular weight of 127 kDa for the Asn832Lysfs*81 and Leu831Ilefs*82 mutations, respectively to 45 kDa for the Ser404* mutation, and to 10 kDa for the cell line carrying the Gln40* mutation could not be visualized. (B) Administration of the immunization peptide in a 5 × excess to antibody concentration reduced all signals, indicating that the N-terminal antibody recognized ADNP specifically in LCLs alongside non-specific band signals. (C-E) C-terminal antibodies detected wild-type ADNP at a molecular weight of 150 kDa. No mutant ADNP was observed with the antibody of Protein Technology which is capable to recognize a part of the truncated Asn832Lysfs*81 and Leu831Ilefs*82 mutations. (F) All C-terminal antibodies visualized wild-type ADNP at 150 kDa, with only the Abcam (p = 0.04; *) and Sarma Laboratory (p = 0.02; *) antibodies showing the expected reduction of ADNP in LCLs of Helsmoortel-Van der Aa syndrome patients. GAPDH was used as a loading control.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Mutagenesis, Western Blot, Control, Expressing, Molecular Weight, Concentration Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 11. Wild-type and mutant ADNP enrichment through immunoprecipitation. The N-terminal sc-F5 ADNP IP-competent antibody was crosslinked to agarose beads and sequentially eluted in fractions (input; flow-through; three consecutive washes, W1-W3; and the immunoprecipitated fracted. IgG non-reactive beads were used as a negative control. In each lane, 20 μg of protein was separated by SDS-PAGE electrophoresis. GAPDH has been used as loading control for all western blots, and critical assessment of the accuracy of the IP method. (A) Immunoprecipitation assay of recombinant wild-type (WT) ADNP and truncating mutants (p.Tyr719*; p.Arg730*; p.Asn832Lysfs*81) in HEK293T overexpression lysates. (B) Immunoprecipitation assay of native wild-type (WT) ADNP and truncating mutants in protein extracts of LCLs derived from a control subject (CTR) and patients with the p.Ser404*, p.Leu831Ilefs*82, or p.Asn832Lysfs*81 ADNP mutation.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Mutagenesis, Immunoprecipitation, Negative Control, SDS Page, Electrophoresis, Control, Western Blot, Recombinant, Over Expression, Derivative Assay

Journal: Separations

Article Title: Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations

doi: 10.3390/separations8110204

Figure Lengend Snippet: Figure 6. The protein pattern and biomarker of the exosomes isolated from different sample volumes. A protein ladder reference (Vivantis Technologies, Shah Alam, Selangor Darul Ehsan, Malaysia, cat no. PR0623) had 2 reference bands (25 and 72 kDa) coupled with blue chromophore for easy identification (A). SDS-PAGE stained with Coomassie blue G250 showed multiple bands with the same patterns (B). The Western blot results showed a single band of TSG101 at about 48.9 kDa exhibiting the exosome biomarker (C). Thirty micrograms of protein were loaded for both SDS-PAGE and the Western blot. All experiments above were run with a reducing agent. M = Marker of protein molecular weight.

Article Snippet: The primary antibodies, human ApoA-1 antibody (cat no. MAB 36641-SP) and human TSG101 antibody (cat no. orb337267), were supplied by R&D System, Inc., Minneapolis, MN, USA and Biorbyt, Cambridge, Cambridgeshire, UK, respectively.

Techniques: Biomarker Discovery, Isolation, SDS Page, Staining, Western Blot, Marker, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy

doi: 10.3390/ijms22031105

Figure Lengend Snippet: ( A ) Western Blot analysis of proteins present (Hsp90, CD63 and TSG101) or absent (calnexin) in exosomes and abundant in bovine milk (β-casein). Protein evaluation in: bovine skimmed milk (SM); exosomes obtained from SM by one (1U) or two (2U) ultracentrifugation steps; and the cellular fraction (CF). Equal amount of protein was loaded. Elution protein profile (F.1, F.8 to F.40) of bovine exosomes isolated by 1U followed by SEC (1U + SEC) ( B ) or 2U + SEC ( C ). Protein concentration (mg/mL) was estimated by the BCA assay. WB of SEC elution fractions from exosomes isolated by 1U + SEC ( D ) or 2U + SEC ( E ). Evaluation of Hsp90, CD63, TSG101, Calnexin and β-casein levels in each fraction (F.1, F.8 to F.40). Mimic hsa-miRNA-148a-3p elution profile (relative expression) of exosomes isolated from skimmed milk by U + T + SEC ( F ) or U + T + U + SEC ( G ). Mw: Molecular weight marker (Bio-Rad).

Article Snippet: Membranes were then incubated with appropriate primary antibodies: anti-Hsp90 (610418, BD, Madrid, Spain), anti-CD63 (bs-1523R, BIOSS, Woburn, MA, USA), anti-TSG101 (A303-506A, Bethyl, Montgomery, TX, USA), anti-calnexin (A303-694A, Bethyl) or anti-β-casein (ab112595, abcam, Cambridge, UK) at 4 °C (overnight).

Techniques: Western Blot, Isolation, Protein Concentration, BIA-KA, Expressing, Molecular Weight, Marker

Journal: Scientific reports

Article Title: ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

doi: 10.1038/s41598-023-51082-4

Figure Lengend Snippet: Figure 3. ROR2 negatively regulates the differentiation of hiPSCs into NS/PCs. (a) Microarray data of ROR2 expression in 10 hiPSC lines (n = 6, biological replicates). Using one-way ANOVA, a significant difference in ROR2 expression was observed between cell lines (P < 0.0001). (b) ROR2 KD was confirmed using qRT-PCR analysis (n = 3, biological replicates). (c) Western blotting analysis of the total extracts obtained from control and ROR2 KD cells. β-actin was used as a loading control. Molecular weight is indicated as Mr (k). (d) qRT-PCR analysis of undifferentiated hPSC markers, OCT3/4 and LIN28A. Total RNA was isolated from R-2A ROR2 KD cells and R-2A control shRNA cells in the undifferentiated state (n = 3, biological replicates). (e, f) qRT-PCR analysis of NS/PC and astrocyte marker genes in NS/PCs derived from ROR2 KD and control shRNA cells (n = 3, biological replicates). Suspension method (e) and adhesion method (f) are shown. (g) Immunofluorescence staining of PAX6 (red) and DAPI (blue) in control (upper) and ROR2 KD (lower) cells. Scale bars, 100 µm. *P < 0.05, **P < 0.01, ****P < 0.0001 (two-tailed unpaired t-test). Error bars represent mean ± SD.

Article Snippet: Primary antibody dilutions were prepared in Can Get Signal immunoreaction enhancer solution (TOYOBO) as follows: anti-ROR2 antibody (AF2064; R&D Systems) 1:1000, anti-β-actin antibody (A5441; Sigma-Aldrich) 1:2000.

Techniques: Microarray, Expressing, Quantitative RT-PCR, Western Blot, Control, Molecular Weight, Isolation, shRNA, Marker, Derivative Assay, Suspension, Immunofluorescence, Staining, Two Tailed Test

Journal: Scientific reports

Article Title: ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

doi: 10.1038/s41598-023-51082-4

Figure Lengend Snippet: Figure 5. ROR2 knockdown promotes differentiation into forebrain neurons. (a) Schematic of culture procedures for midbrain neuron differentiation. (b) qRT-PCR analysis of the mRNA levels of the midbrain neuron markers TH, FOXA2, BN1, and mature neuron marker TUBB3. Total RNA was isolated from R-2A ROR2 KD cells and R-2A control shRNA cells that were differentiated into midbrain neurons (day 42, n = 3, biological replicates). (c) Schematic of culture procedures for forebrain neuron differentiation. (d) qRT-PCR analysis of the mRNA levels of the forebrain neuron markers MAP2, GAD1, SLC6A1, and VGLUT1 and the mature neuron marker TUBB3. Total RNA was isolated from R-2A ROR2 KD cells and R-2A control shRNA cells that were differentiated into forebrain neurons (day 42, n = 3, biological replicates). (e) Immunofluorescence staining of MAP2 (left) and GAD1 (right) in control (upper) and ROR2 KD (lower) cell-derived forebrain neuron. Scale bars, 100 µm. *P < 0.05, **P < 0.01, ****P < 0.0001 (two-tailed unpaired t-test). Error bars represent mean ± SD.

Article Snippet: Primary antibody dilutions were prepared in Can Get Signal immunoreaction enhancer solution (TOYOBO) as follows: anti-ROR2 antibody (AF2064; R&D Systems) 1:1000, anti-β-actin antibody (A5441; Sigma-Aldrich) 1:2000.

Techniques: Knockdown, Quantitative RT-PCR, Marker, Isolation, Control, shRNA, Immunofluorescence, Staining, Derivative Assay, Two Tailed Test