Journal: bioRxiv

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.1101/255802

Figure Lengend Snippet: A HEK293T cells transiently transfected with human iRhom2-3xHA or UNC93B1-3xHA were stained with 4′,6-diamidino-2-phenylindole (DAPI), blue, to label nuclei, anti-HA to label iRhom2-HA (red), and anti-calnexin to label the ER (green). Scale bar: 10 μm. B List of iRhom2 interaction partners identified in the mass spectrometry screen that have either been reported in this study or in previously ( , , ). P-values from a two-sample t-test in Perseus are listed with p-values >0.05 written in grey. P-values were adjusted for multiple hypothesis testing with the Benjamini-Hochberg correction and are listed under “adjusted p-values” with p-values > 0.05 written in grey. C Lysates and anti-HA immunoprecipitation (HA-IP) from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells stably expressing iRhom2-3xHA (indicated) were immunoblotted for HA and FRMD8. Nonspecific bands are marked with an asterisk.

Article Snippet: Human UNC93B1, human iRhom2 WT , iRhom2 Δ100 , iRhom2 Δ200 , iRhom2 Δ300 , iRhom2 Δ382 were amplified from human UNC93B1 (BC025669.1) and iRhom2 cDNA (NM_024599.2; Origene (SC122961)) by PCR and cloned with an C-terminal 3xHA tag into the lentiviral vector pLEX.puro using Gibson assembly (New England Biolabs) following the manufacturer’s instructions.

Techniques: Transfection, Staining, Mass Spectrometry, Immunoprecipitation, Knock-Out, Stable Transfection, Expressing

Journal: Genes and Immunity

Article Title: Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow transplantation

doi: 10.1038/s41435-021-00122-y

Figure Lengend Snippet: Patient, donor, and healthy volunteers characteristics.

Article Snippet: We purchased the specific probe designed for SNP rs308328 (T>C) (product No. C_2623961_1) and TaqMan genotyping master mix from Applied Biosystems.

Techniques:

Journal: Genes and Immunity

Article Title: Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow transplantation

doi: 10.1038/s41435-021-00122-y

Figure Lengend Snippet: Univariate analysis of the association between UNC93B1 polymorphism and post HSCT outcomes.

Article Snippet: We purchased the specific probe designed for SNP rs308328 (T>C) (product No. C_2623961_1) and TaqMan genotyping master mix from Applied Biosystems.

Techniques:

Journal: Genes and Immunity

Article Title: Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow transplantation

doi: 10.1038/s41435-021-00122-y

Figure Lengend Snippet: The Kaplan–Meier analysis of the overall survival rates ( A ), the progression-free survival rates ( B ), the transplant-related mortality rates ( C ), and relapse rates ( D ) after transplantation according to the donor UNC93B1 rs308328 genotype. The solid lines represent the donor C/C genotype, and the dashed lines represent the donor T/T or C/T genotype.

Article Snippet: We purchased the specific probe designed for SNP rs308328 (T>C) (product No. C_2623961_1) and TaqMan genotyping master mix from Applied Biosystems.

Techniques: Transplantation Assay

Journal: Genes and Immunity

Article Title: Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow transplantation

doi: 10.1038/s41435-021-00122-y

Figure Lengend Snippet: Multivariate analysis of the association between UNC93B1 polymorphism and post HSCT outcomes.

Article Snippet: We purchased the specific probe designed for SNP rs308328 (T>C) (product No. C_2623961_1) and TaqMan genotyping master mix from Applied Biosystems.

Techniques:

Journal: International Immunology

Article Title: Expression of murine Unc93b1 is up-regulated by interferon and estrogen signaling: implications for sex bias in the development of autoimmunity

doi: 10.1093/intimm/dxt015

Figure Lengend Snippet: Steady-state levels of Unc93b1 mRNA and protein depend on the sex of mice and treatment of immune cells with estrogen or type I interferon increases the levels. (A) Total RNA from the indicated purified immune cells isolated from either C57BL/6 male (n = 4) or age-matched female (n = 4) mice was subjected to quantitative real-time PCR using the TaqMan assays specific to the murine Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the Unc93b1 mRNA to β2-microglobulin mRNA). The ratio of mRNA levels in CD3+ cells from male mice is indicated as 1. The error bars represent the standard deviation (*P < 0.05). (B) Total cell extracts from the indicated purified immune cells isolated from either C57BL/6 males (M; n = 4) or age-matched females (F; n = 4) were subjected to immunoblotting using antibodies specific to the indicated proteins. FC, fold change in the Unc93b1 protein levels is indicated. (C) Purified CD11b+ cells isolated from C57BL/6 male (n = 6) or age-matched female (n = 6) mice were either left untreated (control) or treated with IFN-α (1000U ml−1), 17β-estradiol (E2; 10nM) or DHT (10nM) as described in Methods for 18h. After the treatment, total cell extracts containing approximately equal amounts of proteins were subjected to immunoblotting using antibodies specific to the indicated proteins. FC, fold change in the Unc93b1 protein levels is indicated.

Article Snippet: Antibodies to murine Unc93b1 protein were purchased from Abnova (cat# {"type":"entrez-protein","attrs":{"text":"PAB13029","term_id":"1236625722"}} PAB13029 ; Walnut, CA, USA).

Techniques: Purification, Isolation, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Control

Journal: International Immunology

Article Title: Expression of murine Unc93b1 is up-regulated by interferon and estrogen signaling: implications for sex bias in the development of autoimmunity

doi: 10.1093/intimm/dxt015

Figure Lengend Snippet: Estrogen signaling contributes to increases in Unc93b1 mRNA and protein levels. (A) Purified CD11b+ cells isolated from C57BL/6 female mice (n = 4) were either left untreated or treated with 17β-estradiol (E2; 10nM) or DHT (10nM) as described in Methods for 18h. Total RNA was subjected to quantitative real-time PCR using the TaqMan assay specific to the murine Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the Unc93b1 mRNA to β2-microglobulin mRNA). The ratio of mRNA levels in untreated cells is indicated as 1. The error bars represent the standard deviation (**P < 0.005). (B) Total RNA that was isolated from splenic cells derived from the wild-type (NZB × NZW)F1 or age-matched ERα-deficient (NZB × NZW)F1 females was analyzed by quantitative real-time PCR using the TaqMan assay specific to the murine Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the Unc93b1 mRNA to β2-microglobulin mRNA). The ratio of mRNA levels in one of the wild-type females is indicated as 1. The error bars represent the standard deviation. NS, not significant. (C) Purified CD11b+ cells from C57BL/6 female mice (n = 4) were either left untreated or treated with 17β-estradiol (E2; 10nM) for the indicated time (h). Total RNA was subjected to quantitative real-time PCR using the TaqMan assay specific to the murine Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the Unc93b1 mRNA to β2-microglobulin mRNA). The ratio of mRNA levels in untreated cells is indicated as 1. The error bars represent the standard deviation (*P < 0.05; **P < 0.005). (D) Purified CD11b+ cells from C57BL/6 female mice (n = 4) were either left untreated or treated with 17β-estradiol (E2; 10nM) for the indicated time (h). After the treatment, total cell extracts containing approximately equal amounts of proteins were subjected to immunoblotting using antibodies specific to the indicated proteins. FC, fold change in the Unc93b1 protein levels is indicated.

Article Snippet: Antibodies to murine Unc93b1 protein were purchased from Abnova (cat# {"type":"entrez-protein","attrs":{"text":"PAB13029","term_id":"1236625722"}} PAB13029 ; Walnut, CA, USA).

Techniques: Purification, Isolation, Real-time Polymerase Chain Reaction, TaqMan Assay, Standard Deviation, Derivative Assay, Western Blot

Journal: International Immunology

Article Title: Expression of murine Unc93b1 is up-regulated by interferon and estrogen signaling: implications for sex bias in the development of autoimmunity

doi: 10.1093/intimm/dxt015

Figure Lengend Snippet: Activation of interferon signaling up-regulates Unc93b1 expression. (A) Purified B220+ cells isolated from C57BL/6 female mice (n = 4) were either left untreated or treated with IFN-α (1000U ml−1), IFN-β (1000U ml−1) or IFN-γ (10ng ml−1) for 6h. Total RNA was subjected to semi-quantitative PCR using a pair of primers that were specific to the murine Unc93b1 gene. (B) RNA samples that were prepared in panel (A) were also subjected to quantitative real-time PCR using the TaqMan assay specific to the murine Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the Unc93b1 mRNA to β2-microglobulin mRNA). The ratio of mRNA levels in untreated cells is indicated as 1. The error bars represent the standard deviation (*P < 0.05; **P < 0.005). (C) Purified B220+ cells isolated from C57BL/6 female mice (n = 4) were either left untreated or treated with IFN-α (1000U ml−1), IFN-β (1000U ml−1) or IFN-γ (10ng ml−1) for 6h. Total cell extracts containing approximately equal amounts of proteins were subjected to immunoblotting using antibodies specific to the indicated proteins. FC, fold change in levels of Unc93b1 protein is indicated. (D) Total cell extracts from splenic cells derived from the wild-type or STAT1-deficient male or age-matched female mice (n = 2) were analyzed by immunoblotting using antibodies specific to the indicated proteins. FC, fold change in levels of Unc93b1 protein is indicated. (E) Total RNA from splenic cells derived from the wild-type or STAT1-deficient male or age-matched female mice (n = 2) was analyzed by quantitative real-time PCR using the TaqMan assay specific to the murine Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units. The ratio of mRNA levels in the wild-type males is indicated as 1. The error bars represent the standard deviation (NS, not significant; *P < 0.05).

Article Snippet: Antibodies to murine Unc93b1 protein were purchased from Abnova (cat# {"type":"entrez-protein","attrs":{"text":"PAB13029","term_id":"1236625722"}} PAB13029 ; Walnut, CA, USA).

Techniques: Activation Assay, Expressing, Purification, Isolation, Real-time Polymerase Chain Reaction, TaqMan Assay, Standard Deviation, Western Blot, Derivative Assay

Journal: International Immunology

Article Title: Expression of murine Unc93b1 is up-regulated by interferon and estrogen signaling: implications for sex bias in the development of autoimmunity

doi: 10.1093/intimm/dxt015

Figure Lengend Snippet: Lupus-prone B6.Nba2 female mice express increased levels of Unc93b1. (A) Total RNA isolated from splenic cells derived from age-matched C57BL/6J (B6), B6.Nba2-C (C) or B6.Nba2-ABC (ABC) female mice (n = 3 for each strain) was subjected to quantitative real-time PCR using the TaqMan assay specific to the Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units. The ratio of mRNA levels in the B6 females is indicated as 1. The error bars represent the standard deviation (**P < 0.005; ***P < 0.001; NS, not significant). (B) Total cell extracts from splenic cells isolated from age-matched C57BL/6J (B6), B6.Nba2-C (C) or B6.Nba2-ABC (ABC) female mice (n = 3) were subjected to immunoblotting using antibodies specific to the indicated proteins. FC, fold change in levels of Unc93b1 protein is indicated.

Article Snippet: Antibodies to murine Unc93b1 protein were purchased from Abnova (cat# {"type":"entrez-protein","attrs":{"text":"PAB13029","term_id":"1236625722"}} PAB13029 ; Walnut, CA, USA).

Techniques: Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, TaqMan Assay, Standard Deviation, Western Blot

Journal: International Immunology

Article Title: Expression of murine Unc93b1 is up-regulated by interferon and estrogen signaling: implications for sex bias in the development of autoimmunity

doi: 10.1093/intimm/dxt015

Figure Lengend Snippet: p202 protein up-regulates Unc93b1 expression. (A) Total RNA isolated from J774.A1 cells that were stably infected with either control lentivirus (control) or the virus encoding shIfi202 was subjected to quantitative real-time PCR using TaqMan assay for the murine Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units. The ratio of mRNA levels in the control cells is indicated as 1. The error bars represent the standard deviation (*P < 0.05). (B) Total cell extracts from cells described in panel (A) were subjected to immunoblotting using the antibodies indicated. (C) Total cell extracts from cells described in panel (B) were subjected to immunoblotting using antibodies specific to the indicated proteins. (D) Total RNA isolated from RAW264.7 cells that were nucleofected with either an empty pCMV vector or the pCMV-202 plasmid (encoding the p202 protein) was subjected to qPCR using TaqMan assay for the Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units. The ratio of mRNA levels in the pCMV nucleofected cells is indicated as 1. The error bars represent the standard deviation (**P < 0.005). (E) Total cell extracts from cells described in panel (C) were subjected to immunoblotting using the antibodies indicated. (F) Total RNA as indicated in panel (D) was subjected to qPCR using TaqMan assay for the Tlr3 and Tlr9 genes. The ratio of the TLR3 and TLR9 mRNA levels to β2-microglobulin mRNA was calculated in units. The ratio of mRNA levels in the pCMV nucleofected cells is indicated as 1. The error bars represent the standard deviation (**P < 0.005; ***P < 0.001).

Article Snippet: Antibodies to murine Unc93b1 protein were purchased from Abnova (cat# {"type":"entrez-protein","attrs":{"text":"PAB13029","term_id":"1236625722"}} PAB13029 ; Walnut, CA, USA).

Techniques: Expressing, Isolation, Stable Transfection, Infection, Control, Virus, Real-time Polymerase Chain Reaction, TaqMan Assay, Standard Deviation, Western Blot, Plasmid Preparation

Journal: International Immunology

Article Title: Expression of murine Unc93b1 is up-regulated by interferon and estrogen signaling: implications for sex bias in the development of autoimmunity

doi: 10.1093/intimm/dxt015

Figure Lengend Snippet: Estrogen or IFN-α-mediated up-regulation of Unc93b1 expression depends on p202 protein expression. (A) Murine J774.A1 cells that were stably infected with either control lentivirus or the virus encoding shIfi202 were either left untreated or treated with IFN-α (IFN, 1000U ml−1) or estrogen (E2, 10nM) for 14h. After the treatments, total RNA was isolated and was subjected to quantitative real-time PCR using TaqMan assay for the murine Unc93b1 gene. The ratio of the Unc93b1 mRNA levels to β2-microglobulin mRNA was calculated in units. The ratio of mRNA levels in the control cells is indicated as 1. The error bars represent the standard deviation (**P < 0.01; ***P < 0.001; NS, not significant). (B) Murine J774.A1 cells that were stably infected with either control lentivirus or the virus encoding shIfi202 were either left untreated or treated with IFN-α (IFN, 1000U ml−1) or estrogen (E2, 10nM) for 14h. After the treatments, total cell lysates were analyzed by immunoblotting for the indicated proteins. FC, fold change in the Unc93b1 protein levels is indicated.

Article Snippet: Antibodies to murine Unc93b1 protein were purchased from Abnova (cat# {"type":"entrez-protein","attrs":{"text":"PAB13029","term_id":"1236625722"}} PAB13029 ; Walnut, CA, USA).

Techniques: Expressing, Stable Transfection, Infection, Control, Virus, Isolation, Real-time Polymerase Chain Reaction, TaqMan Assay, Standard Deviation, Western Blot

Journal: International Immunology

Article Title: Expression of murine Unc93b1 is up-regulated by interferon and estrogen signaling: implications for sex bias in the development of autoimmunity

doi: 10.1093/intimm/dxt015

Figure Lengend Snippet: Proposed model for the up-regulation of murine Unc93b1 expression by activation of interferon and estrogen signaling in immune cells.

Article Snippet: Antibodies to murine Unc93b1 protein were purchased from Abnova (cat# {"type":"entrez-protein","attrs":{"text":"PAB13029","term_id":"1236625722"}} PAB13029 ; Walnut, CA, USA).

Techniques: Expressing, Activation Assay