Journal: eLife

Article Title: Losing Dnmt3a dependent methylation in inhibitory neurons impairs neural function by a mechanism impacting Rett syndrome

doi: 10.7554/eLife.52981

Figure Lengend Snippet:

Article Snippet: Sectioned were mounted in VECTASHIELD HardSet Antifade Mounting Medium without DAPI (Vector Laboratories H-1400) and let dry overnight at 4°C.

Techniques: Labeling, Protease Inhibitor, Lysis, Plasmid Preparation, Electron Microscopy, Western Blot, Antibody Labeling, Purification, Methylation, Lambda DNA Preparation, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation

doi: 10.3389/fcimb.2018.00235

Figure Lengend Snippet: Eap binding to DNA is affected by the state and type of DNA. Eap (0.5 μg/ml) was co-incubated with DNA samples (100 ng/ml) obtained from different sources, and subsequently attached to OTS silicon prior to AFM imaging. Representative AFM height images of Eap/DNA complexes adhering to OTS silicon from three independent experiments are shown: (A) Phage lambda DNA. (B) Artificial 1.4 kb PCR DNA-product. (C) Pst I-digested plasmid pBR322 (D) Circular pBR322 isolated from E. coli DH5α. (E) Sheared herring sperm DNA. Eap molecules putatively adhering to DNA (white arrows), OTS (yellow arrows), or cross-linking DNA (green arrows) are indicated. DNA molecules displayed in (A–C) , and (E) were aligned to OTS silicon by DNA combing.

Article Snippet: Herring sperm DNA (10164142), phage lambda DNA (D3779), and pBR322 (N3033L) DNA were purchased from Invitrogen (Karlsruhe, Germany), Sigma-Aldrich, and New England Biolabs (Frankfurt, Germany), respectively.

Techniques: Binding Assay, Incubation, Imaging, Lambda DNA Preparation, Plasmid Preparation, Isolation

Journal:

Article Title: KIF4 Mediates Anterograde Translocation and Positioning of Ribosomal Constituents to Axons * S⃞

doi: 10.1074/jbc.M808586200

Figure Lengend Snippet: KIF4 interacts with P0. A, amino acids 103-317 of ribosomal protein P0 were identified using the yeast two-hybrid system by screening KIF4 tail domain against an 18-day-old embryonic mouse brain cDNA library. Results from growth assays detected by the ability of cells to grow on medium lacking tryptophan, leucine, and histidine (left panel) revealed by a β-galactosidase filter assays (right panel) on yeast cotransformed with the bait (pGBKT7) and prey (pACT2) plasmids: Segment 1, negative control bait, lamin C/prey, SV40 large T-antigen; segment 2, positive control bait, murine p53/prey, SV40 large T-antigen; segment 3, KIF4 tail domain control bait, KIF4 tail domain/prey, pACT2 (empty); segment 4, clone 44 bait, KIF4 tail domain/prey, P0-C-terminal domain; segment 5, P0 control domain bait, pGBKT7 (empty)/prey, P0-C-terminal domain. Positives interactions are indicated by their growth on tryptophan-, leucine-, and histidine-deficient medium and by the blue color corresponding to β-galactosidase activity. B, total homogenates (HT), SN, and anti-Myc immunoprecipitates (IP) obtained from CHO cells expressing Myc-tagged KIF4-CT plus HA-tagged P0-CT were immunoblotted with anti-Myc (KIF4-CT) and HA (P0-CT). C, a similar experiment but from cells expressing Myc-tagged KIF4-FL and HA-tagged P0-FL. D, a similar experiment but from cells in which IP was carried out with a P0 polyclonal antibody. E, HT, SN, and anti-Myc IPs obtained from CHO cells expressing Myc-tagged KIF4-ΔCT plus HA-tagged P0-FL were immunoblotted with anti-Myc (KIF4-ΔCT) and HA (P0-FL); note the absence of HA-tagged P0 in the IP. F, HT, SN, and anti-Myc IP obtained from CHO cells expressing Myc-tagged KIF2-FL plus HA-tagged P0-FL were immunoblotted with anti-Myc (KIF2-FL) and HA (P0-FL); note the absence of HA-tagged P0 in the IP. G, KIF4 IP from brain microsomal fractions revealed with KIF4 mAbs and the P0 polyclonal antibody. Note that a significant fraction of P0 is present in the KIF4 IP. H, Western blots showing the distribution of KIF4, P0, ribosomal proteins S6, P1/P2, Staufen (Stau) and L1 in different subcellular fractions; note the presence of KIF4 in the membrane and RSW fractions. I, upper panels, RSW, SN, and anti-KIF4 IPs obtained from embryonic (embryonic day 18) rat brain tissue were immunoblotted with the indicated antibodies; note the presence of P0, ribosomal protein S6 and Staufen in the KIF-IPs. I, lower panels, a similar experiment, but from an IP performed with KIF4 preimmune serum; note the absence of KIF4 and P0 in the IP. J, triple immunofluorescence micrograph showing the distribution of ectopically expressed GFP-KIF4 (green), tyrosinated α-tubulin (red), and F-actin (blue; Phalloidin-Alexa 633) in CHO cells; note the presence of GFP-KIF4 in the cell nucleus and in dot-like structures associated with microtubules and the peripheral actin-rich region (arrows). K-M, confocal images showing the distribution of Myc-tagged KIF4 (red), HA-tagged P0 (green), and tyrosinated α-tubulin (blue) in a CHO cell. Note the presence of KIF4 in the cell nucleus (N, arrows) and the colocalization of KIF4 and P0, which is clearly evident at the peripheral leading edge (short arrows). N, merge image showing the distribution of Myc-tagged KIF4 (red) and tyrosinated α-tubulin (green) in a CHO cell extracted with detergents under microtubule stabilizing conditions prior to fixation. O, the same cell but showing the distribution of HA-tagged P0 (red). P, merge images of the same cell showing the distribution of KIF4 (red) and P0 (green). Both KIF4 and P0 remain associated with the cytoskeleton after detergent extraction and colocalize.

Article Snippet: The KIF4-C-terminal (bp 3250-3789) was cloned using a BamHI/BamHI restriction site in pCMV-Myc and in pGBKT7 (Clontech).

Techniques: cDNA Library Assay, Negative Control, Positive Control, Activity Assay, Expressing, Western Blot, Immunofluorescence

Journal: eLife

Article Title: Human RPA activates BLM’s bidirectional DNA unwinding from a nick

doi: 10.7554/eLife.54098

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pTWIN1 (plasmid) , New England BioLabs , N6951S , Expression of core BLM in E. coli.

Techniques: Recombinant, Lambda DNA Preparation, Plasmid Preparation, Expressing, DNA Purification, Staining, Software