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: The TaqMan assays for the Unc93b1 (Assay Id# Mm00457643_m1), Tlr3 (Assay Id# Mm00628112_m1), Tlr9 (Assay Id# Mm00446193_m1), the endogenous Actb control (cat # 4352933E) and β2-microglobulin (Assay Id# Mm00437762_m1) were purchased from Applied Biosystems and used as suggested by the supplier.

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: The TaqMan assays for the Unc93b1 (Assay Id# Mm00457643_m1), Tlr3 (Assay Id# Mm00628112_m1), Tlr9 (Assay Id# Mm00446193_m1), the endogenous Actb control (cat # 4352933E) and β2-microglobulin (Assay Id# Mm00437762_m1) were purchased from Applied Biosystems and used as suggested by the supplier.

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: The TaqMan assays for the Unc93b1 (Assay Id# Mm00457643_m1), Tlr3 (Assay Id# Mm00628112_m1), Tlr9 (Assay Id# Mm00446193_m1), the endogenous Actb control (cat # 4352933E) and β2-microglobulin (Assay Id# Mm00437762_m1) were purchased from Applied Biosystems and used as suggested by the supplier.

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: The TaqMan assays for the Unc93b1 (Assay Id# Mm00457643_m1), Tlr3 (Assay Id# Mm00628112_m1), Tlr9 (Assay Id# Mm00446193_m1), the endogenous Actb control (cat # 4352933E) and β2-microglobulin (Assay Id# Mm00437762_m1) were purchased from Applied Biosystems and used as suggested by the supplier.

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: The TaqMan assays for the Unc93b1 (Assay Id# Mm00457643_m1), Tlr3 (Assay Id# Mm00628112_m1), Tlr9 (Assay Id# Mm00446193_m1), the endogenous Actb control (cat # 4352933E) and β2-microglobulin (Assay Id# Mm00437762_m1) were purchased from Applied Biosystems and used as suggested by the supplier.

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: The TaqMan assays for the Unc93b1 (Assay Id# Mm00457643_m1), Tlr3 (Assay Id# Mm00628112_m1), Tlr9 (Assay Id# Mm00446193_m1), the endogenous Actb control (cat # 4352933E) and β2-microglobulin (Assay Id# Mm00437762_m1) were purchased from Applied Biosystems and used as suggested by the supplier.

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: The TaqMan assays for the Unc93b1 (Assay Id# Mm00457643_m1), Tlr3 (Assay Id# Mm00628112_m1), Tlr9 (Assay Id# Mm00446193_m1), the endogenous Actb control (cat # 4352933E) and β2-microglobulin (Assay Id# Mm00437762_m1) were purchased from Applied Biosystems and used as suggested by the supplier.

Techniques: Expressing, Activation Assay

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: The Journal of Biological Chemistry

Article Title: The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

doi: 10.1016/j.jbc.2022.101607

Figure Lengend Snippet: UNC93B1 deficie ncy reduces store-operated Ca 2+ entry in HEK-293T cells. A , Western blot of protein lysates from mouse dendritic cells (mDCs) and HEK-293T STIM1 flip-in cells immunoprecipitated with αUNC93B1 antibody and probed with αSTIM1 to detect STIM1-UNC93B1 interaction ( top ) and αUNC93B1 to detect UNC93B1 precipitation ( bottom ). B , Western blot of protein lysates from WT, UNC93B1 −/− (clone 52, UKO), and STIM1/2 −/− / UNC93B1 −/− (clone 30, SUKO) HEK-293T cells probed with αSTIM1, αUNC93B1, and αγ-tubulin antibodies. C , averaged Fluo-8 responses evoked by Ca 2+ readmission to 1 μM Tg-treated WT, SKO, UKO, and SUKO cells. D , peak amplitude of the responses in C . Data are mean ± SD from two independent experiments in triplicate. ∗∗∗∗ p > 0.001, ordinary one-way ANOVA ( p < 0.0001, F = 46.20). E , averaged Fura-2 responses evoked by 1 μM Tg and subsequent Ca 2+ readmission to WT and UKO cells expressing KDEL-RFP or mCherry-UNC93B1. F , peak amplitude of the SOCE responses in E . Data are mean ± SD of 120 cells from three independent experiments. NS; ∗∗∗ p > 0.005, ordinary one-way ANOVA ( p < 0.0001, F = 71.67). G , averaged recordings of Fura-2 fluorescence quenching at the isosbestic wavelength (360 nm) during addition of MnCl 2 (500 μM) to untreated WT and UKO cells. H , statistical evaluation of the Mn 2+ -induced quench rates. Data are mean ± SD of 123 cells from three independent experiments. ∗ p > 0.05, unpaired t test. HEK-293T, human embryonic kidney 293T cell; NS, nonsignificant; RFP, red fluorescent protein; SOCE, store-operated Ca 2+ entry; STIM1, stromal interaction molecule 1; Tg, thapsigargin.

Article Snippet: The UNC93B1-FLAG-MYC construct was obtained from Origene (catalog no.: RC210505).

Techniques: Western Blot, Immunoprecipitation, Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

doi: 10.1016/j.jbc.2022.101607

Figure Lengend Snippet: UNC93B1 deficiency reduces STIM1–ORAI1 interactions. A , representative confocal images of WT and UKO cells stained with DAPI ( blue ) and the Duolink proximity ligation assay using mouse αSTIM1 and rabbit αORAI1 antibodies ( red ) before and 10 min after 1 μM Tg addition. B , numbers of immunoreactive dots per cell revealed by the proximity ligation assay. Data are mean ± SD of 22 images from six samples per condition. NS; ∗∗ p > 0.01, two-way ANOVA (interaction: SS = 96.70, DF = 1, MS = 96.70, F (1,84) = 2.758, p = 0.1005). C , representative TIRF images of WT and UKO cells expressing Orai1-YFP ( green ) and mCherry-STIM1 ( red ) treated with 1 μM Tg for 10 min. D , Manders 1 and 2 colocalization index between Orai1-YFP and mCherry-STIM1 before and after Tg addition. Data are mean ± SD of 36 images from three independent experiments. NS; ∗∗∗ p > 0.005; ∗∗∗∗ p > 0.001, two-way ANOVA (M1 interaction: SS = 0.04521, DF = 1, MS = 0.04521, F (1,79) = 7.136, p = 0.0092; M2 interaction: SS = 0.06389, DF = 1, MS = 0.06389, F (1,79) = 5.191, p = 0.0254). E , time course of Tg-induced changes in mCherry-STIM1 TIRF fluorescence in WT and UKO cells. DAPI, 4′,6-diamidino-2-phenylindole; NS, nonsignificant; STIM1, stromal interaction molecule 1; Tg, thapsigargin; TIRF, total internal reflection fluorescence; UKO, UNC93B1 -deficient cell.

Article Snippet: The UNC93B1-FLAG-MYC construct was obtained from Origene (catalog no.: RC210505).

Techniques: Staining, Proximity Ligation Assay, Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

doi: 10.1016/j.jbc.2022.101607

Figure Lengend Snippet: UNC93B1 facilitates STIM1 translocation without reaching ER–PM junctions. A , representative TIRF images of WT cells expressing STIM1-YFP ( right images ) together with KDEL-RFP, mCherry-UNC93B1, or mCherry-UNC93B1 3d ( left images ) after exposure to 1 μM Tg for 10 min to induce STIM1-YFP clustering. B , time course of 1 μM Tg-induced changes in STIM1-YFP TIRF fluorescence. C , kinetics ( left ) and amplitude ( right ) of the changes in STIM1-YFP fluorescence. Data are mean ± SD of 26 to 69 cells from four independent experiments. NS; ∗ p > 0.05; ∗∗ p > 0.01, ordinary one-way ANOVA (half-maximum F = 15.42, p < 0.0001; maximum F = 5.915, p = 0.0037). D , time course of Tg-induced changes in KDEL-RFP, mCherry-UNC93B1, and mCherry-UNC93B1 3d fluorescence. E , averaged amplitude of the changes in RFP or mCherry fluorescence. Data are mean ± SD of 26 to 69 cells from four independent experiments. ∗∗∗∗ p > 0.001, ordinary one-way ANOVA ( F = 15.61, p < 0.0001). ER, endoplasmic reticulum; NS, nonsignificant; PM, plasma membrane; RFP, red fluorescent protein; STIM1, stromal interaction molecule 1; Tg, thapsigargin; TIRF, total internal reflection fluorescence.

Article Snippet: The UNC93B1-FLAG-MYC construct was obtained from Origene (catalog no.: RC210505).

Techniques: Translocation Assay, Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

doi: 10.1016/j.jbc.2022.101607

Figure Lengend Snippet: UNC93B1 deficiency shifts STIM1 to lighter ER domains. A , left , immunoblot of protein lysates of WT cells subjected to subcellular fractionation (1–11; light to heavy ) through an iodixanol gradient (6%, 9%, 12%, 15%, 18%, 21%, 25%, and 27%), UKO cell lysate is a negative control, probed with αUNC93B1. Right , quantification of UNC93B1 immunoreactivity through fractions 1 to 11. B – D , left , immunoblots of subcellular fractions from WT ( top ) and UKO ( bottom ) cells probed with αSTIM1 ( B ), αGAPDH ( C ), and αCalnexin ( D ) antibodies. Right , quantification of STIM1 ( B ), GAPDH ( C ), and calnexin ( D ) immunoreactivity through fractions 1 to 11. Representative of two biological experiments. ER, endoplasmic reticulum; STIM1, stromal interaction molecule 1; UKO, UNC93B1 -deficient cell.

Article Snippet: The UNC93B1-FLAG-MYC construct was obtained from Origene (catalog no.: RC210505).

Techniques: Western Blot, Fractionation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

doi: 10.1016/j.jbc.2022.101607

Figure Lengend Snippet: UNC93B1 binds to the luminal juxtamembrane region of STIM1. A , scheme showing the relevant domains within STIM1 full-length, STIM1 TM4 (TM domain swapped for TLR4 TM domain), STIM1 1–240 , and STIM1 1–152 . B , immunoblot of input and immunoprecipitated (IP) protein lysates from WT cells expressing UNC93B1-FLAG with KDEL-GFP (1), STIM1-YFP (2), STIM1 TM4 -YFP (3), mCherry-STIM1 1–152 (4), or mCherry-STIM1 1–240 (5). UNC93B1 was immunoprecipitated with αFLAG antibody, and the blots were probed with αSTIM1 (N-term) and αFLAG antibodies. The blue arrows indicate the locations of the expected products, and the red arrows indicate the additional reactivities. Representative of two biological experiments. STIM1, stromal interaction molecule 1; TLR4, toll-like receptor 4; TM, transmembrane; YFP, yellow fluorescent protein.

Article Snippet: The UNC93B1-FLAG-MYC construct was obtained from Origene (catalog no.: RC210505).

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

doi: 10.1016/j.jbc.2022.101607

Figure Lengend Snippet: UNC93B1 increases the formation of resting STIM1 dimers. A , scheme showing the STIM1 activation steps tested in the cross-linking assay. STIM1 activation initiated by the unbinding of Ca 2+ ions from luminal EF-hand domains induces the sequential apposition of STIM1 TM and proximal cytosolic helixes. This zipping mechanism can be revealed by crosslinking cysteine-less STIM1 constructs bearing a single cysteine positioned in the TM domain (1-A230C), proximal cytosolic helix (2-M241C), or second cytosolic helix (3-L251C). B , immunoblot analysis of protein lysates from SUKO cells expressing the single cysteine STIM1 constructs with or without UNC93B1-FLAG and treated or not with Tg (0.5 μM for 5 min). Cells were subjected to in vivo crosslinking with aldrithiol-4 and to electrophoresis under nonreducing conditions. Blots were probed with αSTIM1 and αFLAG antibodies. Representative of three independent experiments. C , quantification of the STIM1 dimer ratio from the in vivo crosslink immunoblots. Data are mean ± SD of three experiments. Nonsignificant where there is no indication, ∗ p < 0.05; ∗∗ p < 0.01, unpaired t test. STIM1, stromal interaction molecule 1; Tg, thapsigargin; TM, transmembrane.

Article Snippet: The UNC93B1-FLAG-MYC construct was obtained from Origene (catalog no.: RC210505).

Techniques: Activation Assay, Construct, Western Blot, Expressing, In Vivo, Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

doi: 10.1016/j.jbc.2022.101607

Figure Lengend Snippet: Graphical model. Graphical representation of the role of UNC93B1 as a chaperone that facilitates the early steps of STIM1 activation following ER Ca 2+ depletion. UNC93B1 binds to STIM1 at rest and promotes the initial conformational step of its activation cascade, namely the zipping of the TM and proximal coiled-coil domains ( dark blue bars ) that cause STIM1 to adopt an extended conformation with an exposed channel-activating domain ( red ovals ) that efficiently traps and activates Orai1 channels ( green ) at the PM (WT, left panel ). In the absence of UNC93B1, the maintenance of optimal STIM1–Orai1 coupling is lost ( UNC93B1 −/− , right panel ). ER, endoplasmic reticulum; PM, plasma membrane; STIM1, stromal interaction molecule 1; TM, transmembrane.

Article Snippet: The UNC93B1-FLAG-MYC construct was obtained from Origene (catalog no.: RC210505).

Techniques: Activation 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: 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: