Journal: bioRxiv

Article Title: An essential checkpoint for TLR9 signaling is release from Unc93b1 in endosomes

doi: 10.1101/410092

Figure Lengend Snippet: ( a ) Unc93b1 SKN and Unc93b1 S282A attenuate ligand binding of TLR9. Streptavidin immunoprecipitation of lysates from RAW macrophage lines, expressing TLR9-HA and the indicated Unc93b1 alleles, stimulated for 4h with biotinylated CpG-B (1μM) followed by immunoblot for TLR9. Bar graph shows quantification of CpG-bound TLR9 over total TLR9 from several experiments. ( b ) Unc93b1 SKN cells show normal DNA uptake. Uptake of Cy3-labeled CpG-B (1μM) as measured by flow cytometry of RAW macrophage lines expressing the indicated alleles of Unc93b1. ( c ) Unc93b1 SKN and Unc93b1 S282A bind stronger to TLR9. Flag immunoprecipitation of Unc93b1 from RAW macrophage lines, expressing TLR9-HA and the indicated Unc93b1 alleles, followed by immunoblot of TLR9. Input levels of TLR9 in whole cell lysates (WCL) are also shown. Bar graph shows quantification of TLR9 bound to Unc93b1 from several experiments. ( d ) HA immunoprecipitation of TLR9 from phagosome preparations of RAW macrophage lines, expressing TLR9-HA and the indicated Unc93b1 alleles, followed by immunoblot for Unc93b1. Input levels of TLR9 and Unc93b1 in phagosomes and whole cell lysates (WCL) are also shown; FL: full-length. Representative of two independent experiments. All data are mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t-test. The data are representative of at least three independent experiments, unless otherwise noted.

Article Snippet: Streptavidin Magnetic Beads for biotin pull downs and Protein G agarose were purchased from Pierce.

Techniques: Ligand Binding Assay, Immunoprecipitation, Expressing, Western Blot, Labeling, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

doi: 10.1083/jcb.200805092

Figure Lengend Snippet: Sepharose bead proteome: other proteins of additional classes

Article Snippet: For the endogenous SMN immunoaffinity experiment and the bead proteome experiment comparing Protein G–Agarose (GE Healthcare), Protein G–Sepharose (GE Healthcare), and the magnetic Protein G–Dynabeads (Invitrogen), equivalent total protein amounts of extracts were incubated separately on the appropriate matrices and combined carefully after one wash step in RIPA buffer.

Techniques: Activation Assay, Binding Assay, RNA Binding Assay, Transduction, Migration

Journal: The Journal of Cell Biology

Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

doi: 10.1083/jcb.200805092

Figure Lengend Snippet: Comparison of bead proteomes. (A) Design of the SILAC immunoprecipitation experiment used to compare the bead proteomes of agarose, Sepharose, and magnetic beads. For all three, the protein G–conjugated versions were used. The experiment was performed in two stages, first with a short incubation time of 30 min and next with a long incubation time of 18 h. In addition, cells were fractionated into cytoplasmic and nuclear extracts to compare the profiles of the proteins that bind nonspecifically to the bead matrices. In the case of nuclear extracts, more proteins bind nonspecifically during a long incubation than a short incubation, as assessed both by Coomassie staining (B) and by mass spectrometric analysis (C). The cytoplasmic protein profile did not vary to the same extent. The distribution of proteins by class was quite similar regardless of the cellular extract used in the experiment or the time of incubation (C). Distinct differences in the distribution of these classes of proteins were observed, however, with magnetic beads binding more cytoskeletal and structural proteins nonspecifically and Sepharose binding more nucleic acid binding factors nonspecifically.

Article Snippet: For the endogenous SMN immunoaffinity experiment and the bead proteome experiment comparing Protein G–Agarose (GE Healthcare), Protein G–Sepharose (GE Healthcare), and the magnetic Protein G–Dynabeads (Invitrogen), equivalent total protein amounts of extracts were incubated separately on the appropriate matrices and combined carefully after one wash step in RIPA buffer.

Techniques: Immunoprecipitation, Magnetic Beads, Incubation, Staining, Binding Assay