polyclonal rabbit anti ugt8  (Proteintech)

Journal: Viruses

Article Title: Sulfatide Binds to Influenza B Virus and Enhances Viral Replication

doi: 10.3390/v17040530

Figure Lengend Snippet: Inhibition of sulfatide biosynthesis impairs IBV replication and viral ribonucleoprotein transport. UGT8 inhibitor 19, an inhibitor of galactosylceramide synthase, was used to inhibit sulfatide biosynthesis. Cells were pretreated with 200 nM UGT8 inhibitor 19 for 72 h prior to infection. ( A ) Quantification of B/Lee/1940 infectivity in A549 cells following inhibition of sulfatide synthesis. Infected cells were counted 24 h post-infection and expressed as infected cells/mm 2 . ( B ) Analysis of IBV replication in A549 cells with inhibited sulfatide synthesis, as measured using focus-forming assays. ( C , D ) Immunofluorescence analysis of nuclear export of viral ribonucleoprotein (vRNP). A549 cells infected with B/Lee/1940 were immunostained for nuclei (DAPI, blue) and vRNP (anti-IBV NP followed by Alexa Fluor 555-conjugated anti-mouse IgG; red). White arrows indicate cells with impaired vRNP nuclear export. ( C ) Effects of the ERK signaling inhibitor U0126 on vRNP nuclear export. U0126 was used to confirm the role of ERK signaling in vRNP nuclear export. ( D ) Impact of UGT8 inhibitor and leptomycin B on vRNP nuclear export. Leptomycin B, a nuclear export inhibitor, was used as the positive control. The scale bar represents 40 µm. Data were obtained from three independent experiments for ( A , B ). Values are presented as the mean ± SD. * p < 0.05.

Article Snippet: UGT8 inhibitor 19 [ ] and leptomycin B were purchased from Cayman Chemical (Ann Arbor, MI, USA) and dissolved in DMSO and an ethanol solution, respectively.

Techniques: Inhibition, Infection, Immunofluorescence, Positive Control

Journal: Journal of Biochemistry

Article Title: Characterization of UGT8 as a monogalactosyl diacylglycerol synthase in mammals

doi: 10.1093/jb/mvae084

Figure Lengend Snippet: Exogenous expression of mouse and human UGT8 increased the MGDG content in HeLa cells. (A, B) Immunoblot analysis of HeLa cells transiently expressing N- or C-terminally FLAG-tagged mouse Ugt8. The indicated antibodies were used. (C) The predicted cleavage site of UGT8 (upper panel). AXA (Alanine—X—Alanine) is a canonical motif recognized by signal peptidase, which is conserved in human, mouse and rat UGT8 (lower). (D) Immunoblot analysis of HeLa cells transiently expressing C-terminal FLAG-tagged mouse or human UGT8. Indicated antibodies were used. (E–G) Lipid analysis of HeLa cells transiently expressing C-terminally FLAG-tagged mouse or human UGT8. The MGDG (E) and HexCer (F) contents and the fatty acid composition of MGDG (G) are shown ( n = 3). Mean ± SD. **** P < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test (E–G).

Article Snippet: The UGT8 inhibitor (compound 19) and SCD1 inhibitor (A939572) were purchased from Cayman Chemicals.

Techniques: Expressing, Western Blot

Journal: Journal of Biochemistry

Article Title: Characterization of UGT8 as a monogalactosyl diacylglycerol synthase in mammals

doi: 10.1093/jb/mvae084

Figure Lengend Snippet: UGT8 inhibition decreased the MDGD content in HeLa cells. (A) Immunoblot analysis of HeLa cells treated with control or UGT8 siRNA for 72 h. Indicated antibodies were used. (B–D) Lipid analysis of HeLa cells treated with control or UGT8 siRNA for 72 h. The MGDG (B) and HexCer (C) contents and fatty acid composition of MGDG (D) are shown ( n = 3). (E) Immunoblot analysis of HeLa cells treated with indicated concentrations of UGT8 inhibitor (compound 19) for 24 h. Indicated antibodies were used. (F–H) Lipid analysis of HeLa cells treated with indicated concentrations of UGT8 inhibitor (compound 19) for 24 h. The MGDG (F) and HexCer (G) contents and fatty acid composition of MGDG (H) are shown ( n = 3). Mean ± SD. * P < 0.05, *** P < 0.001, **** P < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test (B, C, F and G). * Cross reaction.

Article Snippet: The UGT8 inhibitor (compound 19) and SCD1 inhibitor (A939572) were purchased from Cayman Chemicals.

Techniques: Inhibition, Western Blot, Control

Journal: Journal of Biochemistry

Article Title: Characterization of UGT8 as a monogalactosyl diacylglycerol synthase in mammals

doi: 10.1093/jb/mvae084

Figure Lengend Snippet: Cellular MGDG was absent in UGT8 KO HeLa cells. (A) Immunoblot analysis of WT or UGT8 KO HeLa cells. Indicated antibodies were used. (B–D) Lipid analysis of WT or UGT8 KO HeLa cells. The MGDG (B) and HexCer (C) contents and fatty acid composition of MGDG (D) are shown ( n = 3). (E, F) Lipid analysis of UGT8 KO HeLa cells (#1) transiently expressing C-terminal FLAG-tagged mouse UGT8 (WT, H358A or H383Q). The MGDG (E) and HexCer (F) contents are shown ( n = 3). (G) Immunoblot analysis of UGT8 KO HeLa cells (#1) transiently expressing C-terminal FLAG-tagged mouse UGT8 (WT, H358A or H383Q). Indicated antibodies were used. Mean ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns; not significant, one-way ANOVA with Dunnett’s multiple comparisons test (B, C, E and F). * Cross reaction.

Article Snippet: The UGT8 inhibitor (compound 19) and SCD1 inhibitor (A939572) were purchased from Cayman Chemicals.

Techniques: Western Blot, Expressing

Journal: Journal of Biochemistry

Article Title: Characterization of UGT8 as a monogalactosyl diacylglycerol synthase in mammals

doi: 10.1093/jb/mvae084

Figure Lengend Snippet: Characterization of the enzymatic activity of UGT8 in vitro. (A–F) Galactosyltransferase activity assay was conducted by incubating 1 mM UDP-Gal and 20 μM DG (10:0/10:0) (A and D) or C2 Ceramide (C2Cer) (B and E) in the presence of 10 μg of HeLa cell lysates expressing mUGT8 with or without UGT8 inhibitor (10 nM) for 1 h at 37°C ( n = 3). For PNGase or CIAP treatment, mUGT8-overexpressed HeLa cell lysates were incubated with PNGase or CIAP for 30 min at 37°C prior to the assay. Exogenous expressions of UGT8 and the effect of PNGase or CIAP treatment were confirmed using either normal or phos-tag SDS-PAGE and immunoblotting (C and F). (G–I) Galactosyltransferase activity assay was conducted by incubating 1 mM UDP-Gal and 20 μM DG (10:0/10:0) (G) or C2 Ceramide (C2Cer) (H) in the presence of 10 μg of UGT8 KO (#1) HeLa cell lysates expressing indicated mUGT8 proteins ( n = 3). Exogenous expressions of UGT8 proteins were confirmed using immunoblotting (I). Mean ± SD. *** P < 0.001, **** P < 0.0001, ns; not significant, two-tailed unpaired t -test (D, E), one-way ANOVA with Dunnett’s multiple comparisons test (A, B, G, H).

Article Snippet: The UGT8 inhibitor (compound 19) and SCD1 inhibitor (A939572) were purchased from Cayman Chemicals.

Techniques: Activity Assay, In Vitro, Expressing, Incubation, SDS Page, Western Blot, Two Tailed Test

Journal: Journal of Biochemistry

Article Title: Characterization of UGT8 as a monogalactosyl diacylglycerol synthase in mammals

doi: 10.1093/jb/mvae084

Figure Lengend Snippet: Membrane lipid saturation-induced UPR was suppressed in UGT8 KO HeLa cells. (A) Immunofluorescence of HeLa cells transiently expressing C-terminal FLAG-tagged mUgt8. Cells were immunostained with indicated antibodies. Scale bar, 10 μm. (B, C) The mRNA level of CHOP in WT or UGT8 KO (#1) HeLa cells treated with SCD1 inhibitor (100 nM) for 24 h (B) or tunicamycin (2.5 μg/ml) for 6 h (C) was quantified using qRT-PCR ( n = 3). (D–G) PERK phosphorylation of WT or UGT8 KO (#1) HeLa cells treated with SCD1 inhibitor (100 nM) for 24 h (D, E) or tunicamycin (2.5 μg/ml) for 6 h (F, G) were evaluated using immunoblotting (representative data of n = 2 out of n = 3 independent experiments). The ratio of phosphorylated PERK (pPERK) to total PERK was quantified (E, G) ( n = 3). (H–J) WT, UGT8 KO (#1) and UGT8 KO (#1) stably expressing mUgt8(WT)-FLAG or mUgt8(H358A)-FLAG HeLa cells were treated with SCD1 inhibitor (100 nM) for 24 h. PERK phosphorylation was evaluated using immunoblotting (F) (representative data of n = 2 out of n = 3 independent experiments). The ratio of phosphorylated PERK (pPERK) to total PERK was quantified (I) ( n = 3). The mRNA level of CHOP was quantified using qRT-PCR (J) ( n = 3). Mean ± SD. * P < 0.05, *** P < 0.001, **** P < 0.0001, ns; not significant, two-tailed unpaired t -test (B, C, E, G), one-way ANOVA with Dunnett’s multiple comparisons test (I, J).

Article Snippet: The UGT8 inhibitor (compound 19) and SCD1 inhibitor (A939572) were purchased from Cayman Chemicals.

Techniques: Membrane, Immunofluorescence, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Stable Transfection, Two Tailed Test

Journal: Molecular Oncology

Article Title: Consensus molecular subtyping of colorectal carcinoma brain metastases reveals a metabolic signature associated with poor patient survival

doi: 10.1002/1878-0261.13748

Figure Lengend Snippet: The identified metabolic gene signature is superior to consensus molecular subtype (CMS) in predicting survival of colorectal cancer (CRC) patients with brain metastasis. Overall survival (OS) of CRC patients suffering from liver or brain metastases stratified according to the CMS classification of the metastatic tissue. Survival impacts were compared between (A) all four subtypes (CMS1 vs. CMS2 vs. CMS3 vs. CMS4), (B) single subtypes and all other subtypes (CMS4 vs. others for CRC liver, CMS3 vs. others for CRC brain), and (C) high and low expression of the seven metabolic signature genes CHAC2 , CA8 , PIK3R3 , CYP26B1 , DHRS9 , UGT8 , and RET within brain metastases. An optimal cut‐off value was calculated to define high/low groups based on the transcriptomic data from metastatic samples. Survival data is shown as Kaplan–Meier curves and differences were tested with a log‐rank test. Hazard ratios with 95% confidence interval and corresponding P ‐values are indicated for all comparisons between two cohorts.

Article Snippet: Shortly, after deparaffinization and peroxidase activity blocking with 5% H 2 O 2 , the samples were exposed to the following primary antibodies: DHRS9 (#14560‐1‐AP), CA8 (#12391‐1‐AP), CYP26B1 (#21555‐1‐AP), PIK3R3 (#27035‐1‐AP), UGT8 (#17982‐1‐AP, all from Proteintech, Rosemont, IL, USA), CHAC2 (#HPA049235; Sigma‐Aldrich, St. Louis, MO, USA) and RET (#ab134100; Abcam, Cambridge, UK).

Techniques: Expressing