Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: The inducibility of IFI27 by IFNα in cell cultures. ( A ) Expression heatmap of significantly upregulated ISGs in HepG2-NTCP cells by IFNα treatment. HepG2-NTCP cells were either left untreated or treated with human IFNα (1,000 IU/mL) for 36 h, followed by mRNA sequencing. Expression values for induced ISGs were Z-score normalized and are displayed as scaled values centered on 0. High and low expressions are indicated in red and blue, respectively. The IFI27 gene is indicated with an arrow. ( B ) Time course analysis of IFI27 mRNA induction by IFNα. HepG2 cells were either left untreated (control) or treated with IFNα (1,000 IU/mL) for 6, 12, 24, and 48 h. The mRNA levels of IFI27 expression were detected by RT-qPCR and normalized to β-actin. ( C ) Dose-dependent induction of IFI27 mRNA by IFNα. PHHs were either left untreated or treated with indicated concentrations of IFNα for 48 h, followed by RT-qPCR analysis of IFI27 mRNA. ( D ) Time-dependent induction of IFI27 protein expression by IFNα. HepG2 cells were treated with IFNα (1,000 IU/mL) and harvested at the indicated time points for Western blot analysis of IFI27 protein levels. β-Actin served as a loading control. Data in the histograms are presented as mean ± SD ( n = 3), *** P < 0.001.

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Sequencing, Control, Quantitative RT-PCR, Western Blot

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: IFI27 inhibits HBV replication primarily through reducing viral RNA. ( A ) HepG2 or ( B ) Huh7 cells were co-transfected with pHBV1.3 plus either a control vector or plasmid FLAG-IFI27. Cells were harvested on day 5 post-transfection, and the levels of intracellular HBV total RNA and cytoplasmic core DNA were analyzed by Northern and Southern blot, respectively. The positions of HBV 3.5 kb pC mRNA and pgRNA as well as 2.4/2.1 kb surface mRNAs on Northern blot were labeled. Ribosomal RNA (rRNA) served as an RNA loading control. On HBV core DNA Southern blot, the positions of the relaxed circular (RC) and the single-stranded (SS) DNAs were indicated. The relative levels of HBV 3.5 kb RNA and single-stranded DNA (ssDNA) in the FLAG-IFI27 overexpression groups were shown as percentages of the control vector groups. The expression of FLAG-IFI27 was analyzed by Western blot. β-Actin served as the protein loading control. ( C ) HepG2 cells were transfected with 20 nM control siRNA (siControl) or IFI27 siRNA (siIFI27) for 16 h, followed by transfection of pHBV1.3 for an additional 3 days. HBV RNA was analyzed by Northern blot. The expression of endogenous IFI27 was analyzed by Western blot. β-Actin served as the protein loading control. The relative levels of HBV 3.5 kb RNA in the siIFI27 group were shown as percentages of the siControl group. ( D ) HepG2-NTCP cells were infected with HBV (500 virion genome equivalents [vge]/cell) for 24 h, followed by transfection with control vector or FLAG-IFI27. Cells continued to be cultured for an additional 5 days. The harvested cells were subjected to HBV total RNA Northern blot, HBV core DNA Southern blot, and FLAG-IFI27 Western blot analyses as described above. β-Actin served as the protein loading control. The relative levels of HBV 3.5 kb RNA and ssDNA are presented as percentages of the control groups. The results shown are representative of at least two experimental trials.

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Southern Blot, Labeling, Over Expression, Expressing, Western Blot, Infection, Cell Culture

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: Subcellular localization of ectopically expressed IFI27 and its effect on cell viability. ( A ) HepG2 cells were transfected with FLAG-IFI27 for 3 days, and the subcellular localization of FLAG-IFI27 was detected by immunofluorescence (green) using anti-FLAG antibodies. The cell nuclei were stained with 4´,6-diamidino-2-phenylindole (DAPI) (blue), and mitochondria were stained with MitoTracker (red). ( B ) HepG2 cells were transfected with control vector, control vector plus pHBV1.3, or pHBV1.3 plus FLAG-IFI27 for 4 days, followed by cell viability measurement by the MTT assay. The relative cell survival rate was normalized to the control vector group (mean ± SD, n = 3; ns: not significant).

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Immunofluorescence, Staining, Control, Plasmid Preparation, MTT Assay

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: IFI27 overexpression inhibits HBV promoter activity. ( A ) Schematic representation of HBV plasmid pHBV1.3 and pCMVHBV. The promoters controlling pgRNA transcription, specifically the HBV EnII/Cp and CMV-IE promoter, are denoted. The nucleotide (nt) positions corresponding to HBV genome (genotype D) at the start and end of the HBV sequence on the plasmids are shown; nt 3182/1 indicates the EcoRI site. The transcription initiation sites of HBV 3.5 kb RNA and 2.4/2.1 kb surface mRNAs are marked; pA indicates the polyadenylation site for all HBV mRNAs. ( B ) HepG2 cells were co-transfected with pHBV1.3 or pCMVHBV plus either a control vector or plasmid FLAG-IFI27 for 3 days, followed by Northern blot analysis of HBV RNA. ( C, D ) HepG2 cells in 96-well plates were co-transfected with 100 ng of each indicated HBV promoter reporter plasmid (EnII/Cp-Luc, S1p-Luc, S2p-Luc), 4 ng of pRL-CMV, plus 100 ng of control vector or FLAG-IFI27, for 3 days, followed by ( C ) Renilla and ( D ) firefly luciferase activity analyses. The Renilla luciferase activity readouts were compared between vector and FLAG-IFI27 groups ( C ). The plotted relative firefly luciferase activities (RLA) of the indicated HBV promoters represent the ratio of luminescence from wells expressing FLAG-IFI27 to that from control vector-transfected wells, normalized to Renilla luciferase as an internal reference ( D ). Data in the histograms are shown as mean ± SD ( n = 3); *** P < 0.001; ns, not significant.

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Activity Assay, Plasmid Preparation, Sequencing, Transfection, Control, Northern Blot, Luciferase, Expressing

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: The endogenous IFI27 plays a role in IFNα-elicited antiviral effect in the HBV infection system. HepG2-NTCP cells were transfected with 20 nM of control siRNA (siControl) or IFI27 siRNA (siIFI27) for 16 h, followed by HBV infection at 500 virion genome equivalents [vge]/cell for 24 h, then the infected cells were left untreated or treated with 1,000 IU/mL of IFN-α for an additional 6 days. The cells were harvested and subjected to the following analyses: ( A ) The IFI27 mRNA knockdown efficiency was verified by RT-qPCR. The relative levels of IFI27 mRNA with or without IFNα treatment are plotted as fold change to the siControl group. ( B ) The IFI27 protein was analyzed by Western blot, and β-Actin served as the protein loading control. HBV total RNA was detected by Northern blot. The relative levels of HBV 3.5 kb mRNA and IFI27 are shown as percentages of the untreated siControl groups. ( C ) HBV pC mRNA was detected by RT-qPCR. The relative HBV pC mRNA levels are presented as fold change compared with the untreated siControl group (mean ± SD, n = 3. ** P < 0.01).

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Infection, Transfection, Control, Knockdown, Quantitative RT-PCR, Western Blot, Northern Blot

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: Assessment of the effect of IFI27 on cellular gene transcription. ( A ) Assessing the effect of IFI27 on mRNA levels of selected cellular transcription factors. HepG2 cells were transfected with the control vector or FLAG-IFI27 for 4 days. The mRNA levels of indicated transcription factors were detected using qPCR with specific primers . The relative mRNA levels of each transcription factor in the FLAG-IFI27 overexpression group are plotted as fold changes compared with the control vector group, following normalization to β-actin mRNA as an internal reference (mean ± SD [ n = 3]). ( B ) HepG2 cells were transfected with either a control vector or FLAG-tagged IFI27 for 4 days, followed by mRNA sequencing and differential gene expression analysis. The volcano plot illustrates the distribution of genes based on log 2 fold change ( x -axis) and –log 10 ( P -value) ( y -axis), representing both the magnitude and statistical significance of expression changes between the comparison. Each point corresponds to a single gene. Red points denote significantly differentially expressed genes (DEGs) with P -values <0.1 and absolute log 2 fold changes >1. Blue points represent genes with P -values <0.1 but log 2 fold changes between –1 and 1. Black points indicate non-significant genes ( P -value ≥0.1). The CXCL8 (interleukin-8 [IL-8]) gene is indicated with an arrow. ( C ) The selected DEGs from ( B ) were validated by RT-qPCR using gene-specific primers . After normalization to β-actin mRNA, the relative DEG mRNA levels in FLAG-IFI27-transfected HepG2 cells are plotted as fold changes versus the control vector group (mean ± SD [ n = 3]; ** P < 0.01, *** P < 0.001). ( D ) Overexpression of IL-8 does not inhibit HBV transcription. HepG2 cells were co-transfected with pHBV1.3 and either a control vector or plasmid pIL-8 expressing human IL-8 for 4 days, followed by HBV RNA Northern blot and IL-8 Western blot analyses. β-Actin served as the protein loading control. ( E ) IL-8 treatment has no effect on HBV transcription. HepG2 cells transfected with pHBV1.3 were either left untreated or treated with recombinant human IL-8 at the indicated concentrations, and the treatment was refreshed every other day over a 4 day period. The cells were then harvested for HBV RNA Northern blot analysis.

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Plasmid Preparation, Over Expression, Sequencing, Gene Expression, Expressing, Comparison, Quantitative RT-PCR, Northern Blot, Western Blot, Recombinant

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: IFI27 reduces the steady-state level of C/EBPα protein. ( A ) IFI27 promotes the protein degradation of C/EBPα. HepG2 cells were co-transfected with control vector or FLAG-IFI27 for 48 h, and then the cells were either left untreated or treated with CHX overnight. The cells were then harvested for protein extraction and Western blot analysis of C/EBPα and FLAG-IFI27. The relative levels of total C/EBPα were quantified and are indicated below the blot. β-Actin served as the protein loading control. ( B ) IFNα induces IFI27 expression and reduces the level of C/EBPα protein. HepG2 cells were left untreated or treated with 1,000 IU/mL of IFNα for 48 h, followed by Western blot analysis of IFI27 and C/EBPα. β-Actin served as the protein loading control. The relative level of total C/EBPα in the IFNα-treated group is shown as a percentage of the untreated group.( C ) IFI27 primarily induces C/EBPα degradation in the cytoplasm. HepG2 cells were transfected with either a control vector or FLAG-IFI27 for 3 days, followed by cell fractionation and Western blot analyses of FLAG-IFI27 and C/EBPα. GAPDH and Lamin A/C served as a marker and loading control for the cytoplasmic and nuclear fractions, respectively. The relative cytoplasmic (Cyto) and nuclear (Nuc) levels of C/EBPα in the FLAG-IFI27 groups are presented as percentages of their respective control groups.

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Plasmid Preparation, Protein Extraction, Western Blot, Expressing, Cell Fractionation, Marker

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: IFI27 suppresses HBV transcription through downregulating C/EBPα. ( A ) IFI27 reduces the enrichment of C/EBPα on HBV genome. HepG2 cells were co-transfected with pHBV1.3 plus either a control vector or FLAG-IFI27 for 3 days. The cells were then subjected to a ChIP assay using ChIP-grade antibodies against H3K27ac (positive control) or C/EBPα, followed by HBV DNA qPCR analysis. The non-immune IgG isotype served as a negative control. The enrichment of IgG, H3K27ac, and C/EBPα on HBV genome is plotted as percentage of input (mean ± SD, n = 3. *** P < 0.001, ns, not significant). ( B ) C/EBPα activates HBV promoters. HepG2 cells in 96-well plates were co-transfected with 100 ng each of HBV promoter reporter plasmids (EnII/Cp-Luc, S1p-Luc, S2p-Luc, and X promoter (Xp)-Luc) and 4 ng of pRL-CMV, plus 100 ng of control vector or plasmid expressing Myc-C/EBPα, for 3 days, followed by dual luciferase assay. The relative luciferase activities are plotted as fold changes (mean ± SD, n = 3. *** P < 0.001). ( C ) C/EBPα overexpression promotes HBV transcription. HepG2 cells were co-transfected with pHBV1.3 plus either a control vector or plasmid Myc-C/EBPα for 3 days. HBV RNA was analyzed by Northern blot. The expression of Myc-C/EBPα was analyzed by Western blot; β-Actin served as the protein loading control. The relative level of HBV 3.5 kb RNA in the Myc-C/EBPα overexpression group is shown as a percentage of the vector control group. ( D ) Depletion of C/EBPα inhibits HBV transcription. HepG2 cells were transfected with 20 nM of siControl or C/EBPα siRNA (siC/EBPα) for 16 h, followed by transfection with pHBV1.3 for an additional 3 days. The cells were then harvested and subjected to Northern blot analysis of HBV RNA. The relative levels of HBV 3.5 kb RNA are presented as percentages of the siControl group. The knockdown efficiency of C/EBPα was confirmed by Western blot. β-Actin served as the protein loading control. ( E ) C/EBPα overexpression abrogates the IFI27-mediated downregulation of HBV transcription. HepG2 cells were co-transfected with pHBV1.3 plus control vector, FLAG-IFI27, or FLAG-IFI27 and Myc-C/EBPα for 3 days. HBV RNA was analyzed by Northern blot. The expression of FLAG-IFI27 and Myc-C/EBPα was analyzed by Western blot; β-Actin served as the protein loading control. The relative levels of HBV 3.5 kb RNA are shown as percentages of the control group.

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Plasmid Preparation, Positive Control, Negative Control, Expressing, Luciferase, Over Expression, Northern Blot, Western Blot, Knockdown

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: IFI27 downregulates C/EBPα through SKP2-mediated ubiquitin-proteasome degradation. ( A ) The IFI27-mediated C/EBPα degradation can be rescued by proteasome inhibitor MG132. HepG2 cells were transfected with control vector or FLAG-IFI27 for 48 h, followed by treatment with 50 µg/mL of CHX in the absence or presence of 5 µM of MG132, as indicated, for an additional 16 h. Cells were then harvested and subjected to Western blot analysis of C/EBPα and FLAG-IFI27. β-Actin served as a loading control. The relative levels of total C/EBPα are presented as percentages of the vector control group. ( B ) IFI27 promotes C/EBPα ubiquitination. HepG2 cells were transfected with Myc-C/EBPα plus control vector or FLAG-IFI27, as indicated, for 48 h, followed by treatment with dimethyl sulfoxide (DMSO) control or 5 µM of MG132 for another 24 h. Cells were then harvested, and the whole-cell lysates were subjected to immunoprecipitation (IP) using antibody against Myc-tag. The immunoprecipitated samples were analyzed by Western blot (WB) using antibodies against ubiquitin, Myc-tag, and FLAG-tag. A fraction (2.5%) of the whole-cell lysates was analyzed by WB as input controls. The relative levels of Myc-C/EBPα are presented as percentages of the vector control group. β-Actin served as the protein loading control. ( C ) Knockdown of SKP2 blocks IFI27-mediated C/EBPα degradation. HepG2 cells were transfected with 20 nM of siControl or SKP2 siRNA (siSKP2) for 24 h, followed by transfection with FLAG-IFI27 or control vector for an additional 48 h. Cells were harvested and subjected to Western blot detection of C/EBPα, SKP2, FLAG-IFI27, and β-Actin. The relative levels of total C/EBPα are presented as percentages of the siControl plus vector control group. ( D ) Depleting SKP2 partially rescued IFI27-mediated downregulation of HBV transcription. HepG2 cells were transfected with 20 nM of siControl or siSKP2 for 16 h, followed by transfection with pHBV1.3 plus control vector or FLAG-IFI27, as indicated, for another 48 h. Cells were then harvested and subjected to Northern blot analysis of HBV RNA and Western blot analysis of C/EBPα, SKP2, FLAG-IFI27, and β-Actin. The relative levels of HBV 3.5 kb RNA and total C/EBPα are presented as percentages of the siControl groups.

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Transfection, Control, Plasmid Preparation, Western Blot, Immunoprecipitation, FLAG-tag, Knockdown, Northern Blot

Journal: Journal of Virology

Article Title: Interferon alpha-inducible protein 27 (IFI27) inhibits hepatitis B virus (HBV) transcription through downregulating cellular transcription factor C/EBPα

doi: 10.1128/jvi.01509-25

Figure Lengend Snippet: The schematic model of the IFI27-mediated antiviral effect on HBV transcription. HBV infects hepatocytes via NTCP-mediated virus entry, followed by cccDNA minichromosome establishment in the cell nucleus, where the cellular transcription factor C/EBPα binds to HBV cccDNA and promotes viral transcription. Upon IFNα treatment, the engagement of IFNα with the heterodimeric interferon alpha receptor 1/2 (IFNAR1/2) activates the intracellular JAK-STAT signaling pathway, leading to induction of ISGs, including IFI27. The upregulation of IFI27 expression in the cytoplasm facilitates the proteasomal degradation of C/EBPα via recruiting an E3 ubiquitin ligase SKP2 for C/EBPα ubiquitination, thereby leading to the downregulation of nuclear levels of C/EBPα and subsequent HBV transcription. See the text for more details. The model figure was created with BioRender.com.

Article Snippet: The siRNA for knocking down IFI27 (Cat# sc-105551), C/EBPα (Cat# sc-37047), SKP2 p45 (Cat# sc-74477), and control siRNA-A (Cat# sc-37007) was purchased from Santa Cruz Biotechnology.

Techniques: Virus, Expressing, Ubiquitin Proteomics

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 1 Mutations of IL27R that enhance the transforming activity of IL27R

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Activity Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 2 Mutant IL27R proteins exhibit enhanced transforming activity compared with IL27R-WT

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Mutagenesis, Activity Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 3 Cells expressing transforming IL27R mutants display increased activation of signalling proteins

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Expressing, Activation Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 4 F523S and F523A mutations of IL27R do not enhance the transforming activity of IL27R-WT

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Activity Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 5 The IL27R- mutant exhibits enhanced homodimeric complex formation

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Mutagenesis

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 6 Comparison of the transforming properties of IL27R proteins to known transforming haemopoietic mutations

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Comparison

Journal: bioRxiv

Article Title: Post-Translational Modifications Remodel Proteome-Wide Ligandability

doi: 10.1101/2025.07.31.667978

Figure Lengend Snippet: a , Distribution of spatial distances between probe-labeled peptides and annotated phosphorylation (left) and N-linked glycosylation (right) sites. b , Proteomic profiling and structural docking reveal ( R )- P6 binding to a site on HSPB1 near S82 (PDB 6VD5). c , STR-induces S82 dephosphorylation in HSPB1. d , Proteomic profile of PTM-dependent liganding of CTSD with ( R )- P6 -labeled peptide (left) and docked structure in identified pocket near S37 (right, AF-P07339-F1). e , STR-induces dephosphorylation at S37 in CTSD. f , P2 PTM-dependent labeling of a site near S87/88 on GABARAPL2 (PDB 7KL3). g , Chemo-precipitation of P2 binding with GABARAPL2 with wild-type and S87/88A mutant GABARAPL2. h , Proteomic profile of PTM-dependent liganding (left) and ChP validation (right) of LAMTOR2 with ( R )- P6 after STR treatment. i, Docked structure of LAMTOR2 with ( R )- P6 (AF-Q9Y2Q5-F1), overlaps with the LAMTOR1 interface in the Ragulator complex (PDB 5X6V). j , STR-induces dephosphorylation at S26 in LAMTOR1. k, Immunoprecipitation (IP) reveals STR-induced reduction in LAMTOR1-LAMTOR2 complex formation. l , Proteomic profile of PTM-dependent liganding of EphA2 with ( S )- P6 -labeled peptide (left) and ChP validation (right) after STR treatment. m , Competitive inhibition of ( S )- P6 binding by ALW-II-41-27, an ATP-competitive EphA2 inhibitor. n , Docking analysis shows ( S )- P6 and ALW-II-41-27 share a binding site near ATP pocket of EphA2 (PDB 5EK7). o , STR-induces Y772 dephosphorylation in EphA2. p , Proteomic profile (left) and ChP validation (right) reveals PTM- and stereo-selective binding of ( R )- P5 to NPC2 with after Tuni treatment. q , Structural docking shows ( R )- P5 binds to the cholesterol-binding pocket of NPC2 near the N58 glycosylation site. r , Dose-dependent competition of ( R )- P5 by cholesterol. Chol., cholesterol. s , Mutation of N58 in NPC2 disrupts ( R )- P5 binding. Phosphorylation experiments were conducted in MDA-MB-231 cells with or without staurosporine treatment (250 nM, 3 h). Glycosylation experiments were conducted with or without tunicamycin (10 µg ml −1 , 24 h) in HEK293T cells. All structures depict probe-labeled residues in dark blue, the remainder of each peptide in light blue, annotated PTM sites in red, functional residues in black, and probes in gold. Proteomic data represent mean ± s.d. from two or three independent biological replicates. Gel images are representative of two independent experiments. Significance was determined using a two-tailed Student’s t -test. Associated datasets are provided in Supplementary Tables 11-12.

Article Snippet: Specific chemical reagents originated from the following sources: staurosporine (Selleckchem, S1421); ALW-II-41-27 (HY-18007), BI-2493 (HY-153723), BI-2865 (HY-153724), AMG-510 (HY-114277), MRTX-849 (HY-130149), RMC-7977 (HY-156498), SHP-099 (HY-100388), dasatinib (HY-10181) are from MedChemExpress; tunicamycin (Merck, T7765); cholesterol (Avanti Research, 700100).

Techniques: Labeling, Phospho-proteomics, Glycoproteomics, Binding Assay, De-Phosphorylation Assay, Mutagenesis, Biomarker Discovery, Immunoprecipitation, Inhibition, Functional Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Post-Translational Modifications Remodel Proteome-Wide Ligandability

doi: 10.1101/2025.07.31.667978

Figure Lengend Snippet: a , Chemical structure of ALW-II-41-27, ATP-competitive EphA2 inhibitor. b , Quantitation of immunoblot validation of EphA2 in . c , Chemo-precipitation (ChP) analysis of probe competition with STR indicating STR does not bind EphA2 competitively. d , Quantitation of immunoblot validation of NPC2 in . e , Chemical structure of NPC2 substrate cholesterol. f , Immunostaining of NPC2 in HEK293T cells with Tuni treatment for 24 h (left) and quantitation (right). Cells were co-stained with LAMP1 as a lysosome marker. Scale bar, 10 μm. Colocalization quantitation were based on cells from six independent fields per condition and presented as mean ± s.d. Immunoblots were quantified from two independent biological replicates and plotted as the mean. Gel images are representative of two independent experiments. Significance was determined using a two-tailed Student’s t -test. n.s., not significant.

Article Snippet: Specific chemical reagents originated from the following sources: staurosporine (Selleckchem, S1421); ALW-II-41-27 (HY-18007), BI-2493 (HY-153723), BI-2865 (HY-153724), AMG-510 (HY-114277), MRTX-849 (HY-130149), RMC-7977 (HY-156498), SHP-099 (HY-100388), dasatinib (HY-10181) are from MedChemExpress; tunicamycin (Merck, T7765); cholesterol (Avanti Research, 700100).

Techniques: Quantitation Assay, Western Blot, Biomarker Discovery, Immunostaining, Staining, Marker, Two Tailed Test