Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-ATG13 , Sigma-Aldrich , Cat# SAB4200100; RRID:AB_10602787.

Techniques: Recombinant, Western Blot, Software

Journal: eLife

Article Title: Inhibition of ULK1/2 and KRAS G12C controls tumor growth in preclinical models of lung cancer

doi: 10.7554/eLife.96992

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-ATG13 (rabbit monoclonal) , Cell Signaling Technology , Cat# 13272 , ELISA (1:500).

Techniques: Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Infection and Immunity

Article Title: Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection

doi: 10.1128/IAI.00887-18

Figure Lengend Snippet: Genetic deletion of B. burgdorferi bbk13. (A) Schematic representation of the linear plasmid 36 (lp36) genomic locus containing bbk13 and adjacent genes. The bbk13 open reading frame (orange arrow) was replaced with the flaBp-aadA antibiotic resistance cassette (black arrow) in wild-type B. burgdorferi (WT) by allelic exchange. Deletion boundaries are indicated by the black dashed lines. Complementation of the bbk13 mutant (Δbbk13) with a wild-type copy of bbk13 under the control of its putative endogenous promoter (the DNA region indicated by the green dotted-line box) on pBSV2G was performed in trans, resulting in the Δbbk13/bbk13+ clone. The bbk13 transcription start site (22) is indicated by a bent black arrow at position 8,903 bp. (B) bbk13 expression is abolished in the Δbbk13 mutant and restored in the Δbbk13/bbk13+ complemented clone. RNA was isolated from WT, Δbbk13, and Δbbk13/bbk13+ clones, and bbk13 expression was measured by RT-qPCR. Gene expression of recA was used for normalization. The mean ± standard deviation from three biological replicates are shown. Statistical significance was tested using one-way ANOVA and Tukey’s multiple-comparison test (*, P < 0.05). (C) The loss of bbk13 does not alter B. burgdorferi growth in vitro. Triplicate cultures of the WT, Δbbk13, and Δbbk13/bbk13+ clones were inoculated at 105 spirochetes/ml. Culture densities were determined every 24 h over a 144-h period using Petroff-Hausser counting chambers. Symbols and error bars represent the mean ± standard deviation.

Article Snippet: The antibodies polyclonal rabbit anti-BBK13 UCF34 (1:10,000), monoclonal mouse anti-FlaB H9724 ( 53 ) (1:200), polyclonal mouse anti-SodA NIH754 (16) (1:1,000), and polyclonal rabbit anti-VlsE (1:1,000) (Rockland Immunochemicals) detected BBK13, FlaB, SodA, and VlsE, respectively.

Techniques: Plasmid Preparation, Mutagenesis, Expressing, Isolation, Clone Assay, Quantitative RT-PCR, Standard Deviation, In Vitro

Journal: Infection and Immunity

Article Title: Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection

doi: 10.1128/IAI.00887-18

Figure Lengend Snippet: Gene bbk13 is constitutively expressed throughout the spirochete’s enzootic cycle and encodes a membrane-associated, immunogenic protein. (A) RT-qPCR using gene-specific primers and a B. burgdorferi genomic standard curve was used to quantify the amount of each target mRNA transcript in infected tick and mouse tissue samples. Copy numbers for each gene target were normalized to recA mRNA copy numbers. Data are presented as the average of biological triplicate samples ± standard deviation. The genes flaB, ospA, and ospC served as controls. ns, not significant by one-way ANOVA with Tukey’s multiple-comparison test. (B) BBK13 protein localization analysis. Total protein lysates (lanes T) of the B. burgdorferi wild-type (WT), Δbbk13, and Δbbk13/bbk13+ clones were fractionated into the soluble (lanes S) and membrane (lanes M) components using ultracentrifugation. Protein fractions from equivalent numbers of spirochetes were subjected to SDS-PAGE and analyzed by immunoblotting with BBK13, periplasm-associated FlaB, or cytoplasm-associated SodA antibodies. (C) Proteinase K susceptibility of BBK13. Equal numbers of B. burgdorferi WT, Δbbk13, and Δbbk13/bbk13+ spirochetes were treated with (+) or without (−) 200 μg/ml proteinase K (PK) in the presence or absence of 0.1% SDS. Samples were subjected to SDS-PAGE and analyzed by immunoblotting with BBK13, periplasm-associated FlaB, or outer surface-associated VlsE antibodies. (D) BBK13 immunogenicity. Recombinant BBK1325–232His protein (BBK13) and B. burgdorferi protein lysate (Bb lysate) were subjected to SDS-PAGE and analyzed by immunoblotting with preimmune and postimmune serum collected from 3 different individual mice before and after infection with B. burgdorferi. Recombinant BBK1325–232His protein (BBK13) was probed with anti-His antibody as a positive control. Protein standards (in kilodaltons) are indicated to the left in panels B, C, and D.

Article Snippet: The antibodies polyclonal rabbit anti-BBK13 UCF34 (1:10,000), monoclonal mouse anti-FlaB H9724 ( 53 ) (1:200), polyclonal mouse anti-SodA NIH754 (16) (1:1,000), and polyclonal rabbit anti-VlsE (1:1,000) (Rockland Immunochemicals) detected BBK13, FlaB, SodA, and VlsE, respectively.

Techniques: Quantitative RT-PCR, Infection, Standard Deviation, Clone Assay, SDS Page, Western Blot, Recombinant, Positive Control

Journal: Infection and Immunity

Article Title: Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection

doi: 10.1128/IAI.00887-18

Figure Lengend Snippet: Loss of bbk13 results in consistently reduced B. burgdorferi loads in distal tissue over multiple time points of infection. Groups of 6 C3H/HeN mice were inoculated intradermally in the dorsal skin with 104 spirochetes of the wild-type (WT), Δbbk13, or Δbbk13/bbk13+ clone. Infection of ear, heart, bladder, and joint tissues was assayed at 2, 3, and 4 weeks postinoculation. (A to C) Tissues collected from groups of 6 mice at 3 weeks postinoculation with B. burgdorferi clones were incubated in BSKII medium, and the spirochete density was scored by dark-field microscopy in a semiquantitative manner, where 0 indicates no spirochetes (white) and 1 to 3 indicate increasing spirochete densities (pink to red shading). (A) Reisolation cultures of tissue from WT-inoculated mice were monitored and scored for spirochete density at various time points of incubation (the age of the reisolation culture) until all cultures of each tissue type attained a score of 3. (B) Reisolation cultures of tissue from Δbbk13 and Δbbk13/bbk13+ spirochete-inoculated mice were scored for density on the time point of incubation when all WT cultures of each tissue type attained a score of 3 (6-day culture). (C) All tissue reisolation cultures were scored for endpoint density (14-day culture). (D) Spirochete loads in mouse ear, heart, and joint tissues were quantified by qPCR at 2, 3, and 4 weeks postinoculation. The data are presented as the number of flaB copies per 106 nid copies. Symbols and error bars represent the mean ± standard deviation. The Δbbk13 mutant loads were below the level of detection in heart tissue at all time points examined (symbols placed on the axis represent tissues that were tested but for which results were are not plottable on a logarithmic axis). For samples sets in which the spirochete loads in some or all of the tissues from the groups of 6 mice were below the level of detection, the number of tissues out of 6 with detectable loads are indicated. Two-way ANOVA and Tukey’s multiple-comparison test were used to test for statistical significance compared to the results for the WT (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Article Snippet: The antibodies polyclonal rabbit anti-BBK13 UCF34 (1:10,000), monoclonal mouse anti-FlaB H9724 ( 53 ) (1:200), polyclonal mouse anti-SodA NIH754 (16) (1:1,000), and polyclonal rabbit anti-VlsE (1:1,000) (Rockland Immunochemicals) detected BBK13, FlaB, SodA, and VlsE, respectively.

Techniques: Infection, Clone Assay, Incubation, Microscopy, Standard Deviation, Mutagenesis

Journal: Infection and Immunity

Article Title: Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection

doi: 10.1128/IAI.00887-18

Figure Lengend Snippet: B. burgdorferi spirochetes lacking bbk13 are found at a reduced density in the blood during dissemination. Blood was collected daily from cohorts of 6 C3H/HeN mice inoculated intradermally with 104 spirochetes of the WT, Δbbk13, or Δbbk13/bbk13+ clone and plated in BSK-agarose for determination of the number of CFU. The data are reported as the number of spirochetes per milliliter of blood. Symbols and error bars represent the mean ± standard deviation. Two-way ANOVA and Tukey’s multiple-comparison test were used to test for statistical significance compared to the results for the WT (****, P < 0.0001; ***, P < 0.001; **, P < 0.01).

Article Snippet: The antibodies polyclonal rabbit anti-BBK13 UCF34 (1:10,000), monoclonal mouse anti-FlaB H9724 ( 53 ) (1:200), polyclonal mouse anti-SodA NIH754 (16) (1:1,000), and polyclonal rabbit anti-VlsE (1:1,000) (Rockland Immunochemicals) detected BBK13, FlaB, SodA, and VlsE, respectively.

Techniques: Standard Deviation

Journal: Infection and Immunity

Article Title: Borrelia burgdorferi bbk13 Is Critical for Spirochete Population Expansion in the Skin during Early Infection

doi: 10.1128/IAI.00887-18

Figure Lengend Snippet: Loss of bbk13 results in deficient spirochete expansion at the skin inoculation site during early infection. Spirochete load kinetics at the skin inoculation site were determined by qPCR at different time points after intradermal inoculation of groups of 6 C3H/HeN mice with 104 spirochetes of the WT, Δbbk13, or Δbbk13/bbk13+ clone, as described in the legend to Fig. 2. The data are presented as the number of flaB copies per 106 nid copies. Early kinetics were determined by quantitating the spirochete loads in the skin inoculation sites on days 0, 1, 3, 5, and 7 postinoculation. Columns and error bars represent the mean ± standard deviation. Two-way ANOVA and Tukey’s multiple-comparison test were used to test for statistical significance compared to the results for the WT (**, P < 0.001).

Article Snippet: The antibodies polyclonal rabbit anti-BBK13 UCF34 (1:10,000), monoclonal mouse anti-FlaB H9724 ( 53 ) (1:200), polyclonal mouse anti-SodA NIH754 (16) (1:1,000), and polyclonal rabbit anti-VlsE (1:1,000) (Rockland Immunochemicals) detected BBK13, FlaB, SodA, and VlsE, respectively.

Techniques: Infection, Standard Deviation