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: