Journal: Journal of Proteomics

Article Title: Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis

doi: 10.1016/j.jprot.2012.06.024

Figure Lengend Snippet: Verification of protein expression and localization in synovial fluid and membrane. A. Representative MALDI-TOF mass spectra of spot 873 identified as vitamin D-binding protein. The matched peptide sequences are underlined within the sequence of vitamin D-binding protein. An approximate molecular weight of the protein cleavage product (between the arrows) is calculated from the amino acid sequence, in line with DIGE estimate. B. Immunoprecipitation of VDBP from synovial fluids of n = 48 representative JIA patients (indicated by number in lanes 1–48; M — molecular weight marker; D — purified human VDBP positive control). Band densities of vitamin D binding protein concur with protein expression levels measured by DIGE (extended-to-be oligoarticular patients in lanes 13, 21, 24, 36, 37, 45, 47, 48). C. Representative immunohistochemistry of neighboring sections of synovial membrane from a polyarticular patient, all captured at 10 × magnification. Vitamin D binding protein expression is perivascular in nature. D. ELISA quantification of c-reactive protein and vitamin D binding protein concentration in initial plasma and synovial samples taken from the whole study cohort. Error bars on the box-whisker plots represent range between maximum and minimum values; center line represents median.

Article Snippet: A purified form of human VDBP was run with IP samples as a positive control (2 μg) (Athens Research & Technology Inc., Athens, GA, USA).

Techniques: Expressing, Membrane, Binding Assay, Sequencing, Molecular Weight, Immunoprecipitation, Marker, Purification, Positive Control, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Protein Concentration, Clinical Proteomics, Whisker Assay

Journal: BMC cell biology

Article Title: Peptide aptamers as new tools to modulate clathrin-mediated internalisation--inhibition of MT1-MMP internalisation.

doi: 10.1186/1471-2121-11-58

Figure Lengend Snippet: Figure 1 Isolation and validation of PAs interacting with MT1-MMP ICD. (A) Schematic representation of the TrxA scaffold and the PAs swiggle, 76, and s14. The sequences of the peptide inserted in TrxA (swiggle, s14) or fused to the N-terminal region of TrxA (76) are detailed. The point mutation in the sequence of s14 is depicted in bold and the amino acids in lower cases in PA 76 represent linkers between the multiple peptides. * denotes the stop codon. (B) Swiggle interacts with the MT1-MMP ICD in a yeast-two hybrid interaction assay. EGY48 cells expressing LexA-DBD, LexA-MT1, LexA-MT2, LexA-MT3, LexA-MT5 or LexA-Cdk4 were mated with EGY42 cells expressing AD-TrxA, AD-swiggle, AD-s14, AD-76 or AD-CyclinD1 and plated onto selective media.

Article Snippet: Oligonucleotides coding for a PGGG linker followed by MT1-MMP, MT2-MMP, MT3-MMP, or MT5-MMP intracellular domain (ICD) were annealed and cloned downstream of the LexA DNA binding domain (DBD) in pEG202 (Origene, Rockville, USA) to generate LexAMT1, -MT2, -MT3 and -MT5.

Techniques: Isolation, Biomarker Discovery, Mutagenesis, Sequencing, Expressing

Journal: BMC cell biology

Article Title: Peptide aptamers as new tools to modulate clathrin-mediated internalisation--inhibition of MT1-MMP internalisation.

doi: 10.1186/1471-2121-11-58

Figure Lengend Snippet: Figure 8 Swiggle interacts with the LLY573 motif of the MT1-MMP ICD. (A) EGY48 cells expressing LexA-DBD, LexA-MT1, the LexA-MT1 LLY/ A mutant or LexA-Cdk4 were mated with EGY42 cells expressing AD-swiggle, AD-s14 or AD-CyclinD1 and plated onto selective media. (B) Cell lysates prepared from MCF7 cells expressing MT1-MMP alone (lanes 1 and 2) or together with GFP-s14 (lane 3) or GFP-swiggle (lane 4) were subjected to immunoprecipitation in the presence (lanes 2, 3 and 4) or the absence (lane 1) of an anti-MT1-MMP pAb The presence of μ2 and MT1-MMP in the immunocomplexes was monitored by Western blotting. The expression level of GFP-s14, GFP-swiggle or μ2 in the input lysates was analysed by Western blot.

Article Snippet: Oligonucleotides coding for a PGGG linker followed by MT1-MMP, MT2-MMP, MT3-MMP, or MT5-MMP intracellular domain (ICD) were annealed and cloned downstream of the LexA DNA binding domain (DBD) in pEG202 (Origene, Rockville, USA) to generate LexAMT1, -MT2, -MT3 and -MT5.

Techniques: Expressing, Mutagenesis, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: BEX1 is an RNA-dependent mediator of cardiomyopathy

doi: 10.1038/s41467-017-02005-1

Figure Lengend Snippet: Annotating the BEX1 protein interactome. a Silver-stained polyacrylamide gel following GST-only or GST-BEX1 pull-down from cardiac protein extracts. Arrows indicate the binding proteins identified by mass-spectrometry. b Silver-stained polyacrylamide gel following control or Flag-BEX1 immunoprecipitation from neonatal rat cardiomyocytes protein extracts. Arrows indicate the binding proteins identified by mass-spectrometry. c Schematic of the employed yeast two-hybrid screening strategy. The arrow shows the list of binding proteins identified. d Western blot following GST-BEX1 pull-down of the indicated proteins from cardiac extracts of WT hearts with or without addition of micrococcal nuclease to degrade RNA. e Western blot for the indicated 5 proteins following immunopreciptation of BEX1 protein from BEX1 transgenic hearts. Input is shown on the left and anti-BEX1 or IgG was used for the immunoprecipitations. f Western blot for BEX1 protein from a BEX1 transgenic heart after immunoprecipitation of the 5 shown endogenous proteins. The input is shown on the bottom and the IgG control is shown in the right lanes. g Immunofluorescence for BEX1 (green) in neonatal rat cardiomyocytes in unstimulated (serum-free media) or hypertrophic conditions (media containing 2% serum). Original magnification is 100×. Scale bar is 20 µm. Abbreviations: DDX3x = DEAD box helicase 3 X-linked; DDX1 = DEAD box helicase 1; hnRNPH1 = heterogeneous nuclear ribonucleoprotein H1; PHF11 = PHD-containing factor 11; HSP70 = heat shock protein 70; TFEB = transcription factor EB; NACA = Nascent Polypeptide-Associated Complex Alpha Subunit; EPRS = Glutamyl-Prolyl-tRNA Synthetase; DNAJB11 = DnaJ Heat Shock Protein Family Member B11; PAFAH1B1 = Platelet-Activating Factor Acetylhydrolase 1b Regulatory Subunit 1; KIF26b = Kinesin Family Member 26b; MAP4 = Microtubule-Associated Protein 4. All the gels show molecular weight markers in kiloDaltons

Article Snippet: Co-immunoprecipitation from mouse hearts was performed using antibodies against BEX1 (kindly provided by Dr. Frank Margolis; rabbit polyclonal; used 1:2000 for visualization by Western Blot), DDX1 (Proteintech 11357-1-AP; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), DDX3x (Bethyl Laboratories A300-474A; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), PHF11 (Millipore ABE396; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), EPRS (Abcam ab31531; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), hnRNPH1 (Abcam ab10374; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), or normal IgG control.

Techniques: Staining, Binding Assay, Mass Spectrometry, Control, Immunoprecipitation, Two Hybrid Screening, Western Blot, Transgenic Assay, Immunofluorescence, Molecular Weight

Journal: Nature Communications

Article Title: BEX1 is an RNA-dependent mediator of cardiomyopathy

doi: 10.1038/s41467-017-02005-1

Figure Lengend Snippet: Summary of BEX1-interacting proteins assembled from 3 different screens that fit into a classification of RNA interacting, transport and maturation

Article Snippet: Co-immunoprecipitation from mouse hearts was performed using antibodies against BEX1 (kindly provided by Dr. Frank Margolis; rabbit polyclonal; used 1:2000 for visualization by Western Blot), DDX1 (Proteintech 11357-1-AP; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), DDX3x (Bethyl Laboratories A300-474A; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), PHF11 (Millipore ABE396; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), EPRS (Abcam ab31531; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), hnRNPH1 (Abcam ab10374; rabbit polyclonal; diluted 1:1000 for visualization by Western Blot), or normal IgG control.

Techniques: