Univariate analysis and multivariate logistic regression analysis of the anxiety in infertile women.

Univariate analysis and multivariate logistic regression analysis of the anxiety in infertile women.</p

The flow diagram of this study.

Abbreviation: GAD-7: Generalized Anxiety Disorder 7; PHQ-15: Patient health questionnaire-15; PSQI: Pittsburgh sleep quality index; PHQ-9: Patient Health Questionaire-9.</p

Univariate analysis and multivariate logistic regression analysis of the depression in infertile women.

Univariate analysis and multivariate logistic regression analysis of the depression in infertile women.</p

The details of four scales and reliability and validity analysis of four scales.

The details of four scales and reliability and validity analysis of four scales.</p

The baseline characteristics of infertility women in this study.

The baseline characteristics of infertility women in this study.</p

Mutation of ubiquitination sites impairs innate antiviral response.

<p>(<b>A</b>) ISRE reporter activity in control, FLAG tagged TBK1, and K→R mutant transfected HEK293 cells. Data present the mean ± SD of triplicate samples. Asterisk indicates P<0.01. (<b>B</b>) ISRE reporter activity of TBK1 K→R mutants. Various TBK1 mutants were transfected into HEK293 cells with ISRE and Renilla reporter plasmids. (<b>C</b>) <i>Tbk1^−/−</i> MEFs were transfected with TBK1 and TBK1 mutants. After 48 hr cells were treated with 1 µg/ml poly(I:C) for the designated times. Relative levels of IFNβ and RANTES RNA are presented. Asterisk indicates P<0.01. (<b>D</b>)<i>Tnfr1</i>^−/−, <i>tbk1</i>^−/− macrophages were transfected with FLAG-tagged TBK1 and TBK1 mutants. After 24 hr cells were infected with 0.1 MOI (multiple of infection) VSV-eGFP for 12 hr. Control TBK1 levels are included in the lower panel. (<b>E</b>) VSV-Luciferase (VSV-Luc) replication in HEK293 cells transfected with TBK1 or TBK1 mutants. Cells were infected with VSV-Luc at 1 MOI. Luciferase reporter activity was measured 18 hr after infection. (<b>F</b>) Induction of IFNβ in <i>tbk1</i>^−/− MEFs transfected with indicated TBK1 constructs. Two days later cells were treated with 5 HA Sendai virus for 12 hr. Data represent the mean ± SD of triplicate samples. *P<0.05 was calculated by comparison to wild type TBK1.</p

Model of TBK1 recruitment to MAVS.

<p>Double-stranded RNA and RNA viruses induce MIB-TBK1 association resulting in K63-linked TBK1 pUb. NEMO binds ubiquitinated TBK1 and the complex is recruited to MAVS. The MAVS signalosome serves as platform for TBK1 activation leading to IRF3 phosphorylation.</p

NEMO bridges TBK1 to MAVS.

<p>(<b>A</b>) IRF3 phosphorylation in Sendai infected wild type and <i>nemo</i>^−/− MEFs. (<b>B</b>) TBK1 ubiquitination in Sendai infected wild type and <i>nemo</i>^−/− MEFs. (<b>C</b>) Reciprocal immunoprecipitation between MAVS and NEMO. NEMO- MYC and MAVS- FLAG were transfected into HEK293 cells after 48 hr cells were collected. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (<b>D</b>) Wild type and <i>nemo</i>^−/− MEFs were cotransfected with FLAG-TBK1 and HA-MAVS after 48 hr cells were infected with 5HA Sendai virus. Cell lysates were immunoprecipitated and immunoblotted with the designated antibodies.</p

Mass spectrometry identifies TBK1 ubiquitination sites in response to RNA virus.

<p>(<b>A</b>) Map of various TBK1 truncation mutants showing the schematic structure of kinase domain, ubiquitin-like domain (ULD), and coiled coil domain (CC). (<b>B</b>) Kinase and ubiquitin-like domains are required for TBK1 activity. FLAG-tagged TBK1 truncation mutants were transfected with Ub-K63-HA into HEK293 cells. After 48 hr cells were collected, immunoprecipitated, and immunoblotted with the specified antibodies. Molecular weights are indicated. (<b>C</b>) ISRE reporter activity of HEK293 cells transfected with indicated TBK1 truncation mutants. (<b>D</b>) TBK1 complexes from control or Sendai infected 293 cells stably transfected with FLAG-TBK1 were affinity purified, then separated on a Nu-PAGE gel which was stained with Commassie blue. The fraction labeled Ub was sent for mass spectrometric analysis. Right panel shows the schematic structure of TBK1 kinase domain (KD), ubiquitin-like domain (ULD), and coiled coil domain (CC). Ubiquitination sites identified by mass spectrometry are indicated by arrowheads and peptides are detailed in the box. (<b>E</b>) HEK293 cells were cotransfected with designated FLAG-tagged TBK1 mutants and HA-tagged K63-only ubiquitin. Lysates were immunoprecipitated and immunoblotted as designated.</p

Ubiquitination regulates TBK1 kinase activity.

<p>(<b>A</b>) IRF3 and TBK1 phosphorylation in <i>tbk1</i>^−/− MEFs transfected with indicated TBK1 constructs. Two days later cells were infected with Sendai virus. (<b>B</b>) <i>In vitro</i> kinase assay of TBK1 and TBK1 mutants using IRF3 as substrate. HEK293 cells were transfected with indicated FLAG-tagged TBK1 constructs after 48 hr TBK1 was purified with anti-FLAG. Purified TBK1 was examined for kinase activity using IRF3 as substrate. (<b>C</b>) <i>Mib1^f/f</i>, <i>mib1</i>^−/−, or <i>mib1</i>^−/− cells transfected with MIB2 siRNA (<i>mib2</i>^KD) were treated with 50 HA Sendai virus for the designated times. Cell lysates were immunoblotted as indicated.</p