Journal: Life Science Alliance

Article Title: Multi-region proteome analysis quantifies spatial heterogeneity of prostate tissue biomarkers

doi: 10.26508/lsa.201800042

Figure Lengend Snippet: Six proteins (ACPP, ABCF1, NUP93, CUTA, CRAT, and FSTL1) were measured using IHC in a TMA containing tissue samples from 83 patients from an independent cohort, including 35 patients with BPH and 48 patients with prostate ADCA.

Article Snippet: The following primary antibodies were used to stain 4-μm slides of the TMA using the Ventana Benchmark (Roche Ventana Medical Systems, Inc.) automated staining system: ACTR1B (1:400; abcam, 60 min pretreatment), Desmin/DES (1:20; Dako A/S, 16 min pretreatment), KLK3/PSA (1: 10000; Dako A/S) and GDF15 (1:50; Biorbyt, 30 min pretreatment), ACPP (1:2000; DAKO A/S), ABCF1 (1:50; Novus Biologicals, 90 min pretreatment), NUP93 (1:50; Novus Biologicals, 60 min pretreatment), CUTA (1:100; Lifespan Biosciences, 60 min pretreatment), CRAT (1:100; Atlas Antibodies, 30 min pretreatment), and FSTL1 (1:100; Atlas Antibodies, 16 min pretreatment).

Techniques:

Journal: bioRxiv

Article Title: RNase L activating 2′–5′ oligoadenylates bind ABCF1, -3 and Decr-1

doi: 10.1101/2023.03.21.532770

Figure Lengend Snippet: (A) Scheme of affinity purification with 2′–5′ OA conjugated beads and human, fly and mouse lysates for mass spectrometry. Cell lysates were incubated with beads linked to ATP or 2′–5′ OA, followed by affinity enrichment and analysis of bead-bound proteins using LC-MS/MS. (B-D) Identification of 2′–5′ OA binding proteins in (A) human (HeLa S3, n = 4), (B) mouse (BMDMs, n = 3) and (D) Drosophila (Schneider S2, n = 4) cell lysates. Volcano plots show the log 2 fold change enrichment of proteins in 2’–5’ OA vs ATP samples (x-axis) plotted against the log 10 transformed p-value (y-axis). Proteins in blue are significantly enriched (two-sided Student’s t-test, S0 = 0, FDR < 0.01, log 2 fold change ≥ 1.5) and proteins in orange are significantly enriched hits belonging to the ABCE and ABCF subfamilies/ABC superfamily. (E, F) Validation of ABCF1, -3 and RNAse L binding to the indicated affinity beads in HeLa (E) and THP-I (F) cell lysates that were treated with type-I IFN overnight. (G) as for (E) but 293T cell lysates from HA-ABCF1 overexpressing cells were used. (H) Binding affinity of recombinant ABCF1 to fluorescent 2′–5′ OA or OH-2′–5′ OA as determined by microscale thermophoresis (MST). The inset shows a coomassie stain on a SDS-PAGE of the recombinant protein. The graph shows mean ± sd for 2′–5′ OA (n = 4) and OH-2′–5′ OA (n = 3). (I) Precipitation of recombinant ABCF1 and the indicated variants of ABCF1 with mutations in walker A and B motifs with 2′–5′ OA and visualization by coomassie stain.

Article Snippet: Antibodies against the following proteins were used: ABCF1 (Aviva Systems biology; ARP43631_P050), ABCF3 (Sigma; HPA036332), RNASE L (Abcam; ab13825), HA tag (HRP coupled, Sigma; H6533), β-actin (HRP coupled, Santa Cruz; sc-47778) and HRP coupled secondary antibodies against rabbit IgG (Cell Signaling Technology; 7074) and against murine IgG (Columbia Biosciences; HRP-112).

Techniques: Affinity Purification, Mass Spectrometry, Incubation, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Transformation Assay, Biomarker Discovery, Recombinant, Microscale Thermophoresis, Staining, SDS Page

Journal: bioRxiv

Article Title: RNase L activating 2′–5′ oligoadenylates bind ABCF1, -3 and Decr-1

doi: 10.1101/2023.03.21.532770

Figure Lengend Snippet: (A) Immunoblot analysis for endogenous RNase L in HeLa and HeLa S3 cells. (B, C) HeLaS3 and HeLa cells were electroporated with siRNA against ABCF1 (siABCF1) or non-targeting control siRNA (siCTRL) and expression levels of ABCF1 and GAPDH mRNA were measured by qPCR. Bar graphs show normalized average expression levels 48 h after siRNA treatment. (D, E) HeLa and HeLa S3 cells from (B, C) were transfected with 300ng polyI;C or mock transfected using Lipofectamine 2000. 24h later total RNA was isolated and visualized on an agarose gel. 28s and 18s rRNA are indicated. (F, G) HeLa S3 cells from (B) were infected with EMCV (MOI: 0.01) (F) or LACV (MOI: 1) (G) and accumulation of virus was titrated on Vero E6 cells 24h after infection. (H, I) as (F, G) but HeLa S3 cells were used. (J) A549 cells were transduced with lentiviral vectors expressing puromycin resistance, Cas9 and gRNAs targeting ABCF1, ABCF3 or non-targeting controls (NEG1 and NEG2) and selected for puromycin resistance. Obtained cells were infected with the indicated viruses expressing fluorescent reporter proteins. The bar graphs show mean fluorescent intensity (± sd, n=4) normalized to cell confluence for timepoint of 24 hpi (VSV-eGFP MOI 0.2, RVFV-Katushka MOI 0.5), 40 hpi (YFV-Venus MOI 2), 48 hpi (HSV-1-mCherry MOI 2) or 72 hpi (VACV-eGFP MOI 0.01). One representative experiment of three is shown.

Article Snippet: Antibodies against the following proteins were used: ABCF1 (Aviva Systems biology; ARP43631_P050), ABCF3 (Sigma; HPA036332), RNASE L (Abcam; ab13825), HA tag (HRP coupled, Sigma; H6533), β-actin (HRP coupled, Santa Cruz; sc-47778) and HRP coupled secondary antibodies against rabbit IgG (Cell Signaling Technology; 7074) and against murine IgG (Columbia Biosciences; HRP-112).

Techniques: Western Blot, Control, Expressing, Transfection, Isolation, Agarose Gel Electrophoresis, Infection, Virus, Transduction

Journal: bioRxiv

Article Title: RNase L activating 2′–5′ oligoadenylates bind ABCF1, -3 and Decr-1

doi: 10.1101/2023.03.21.532770

Figure Lengend Snippet: (A) Experimental scheme of the pulsed SILAC approach used to measure protein synthesis in HeLa and HeLa S3 cells after treatment with OH-2′–5′ OA or 2′–5′ OA. (B) Density scatter plot comparing the normalized and log 2 transformed intensity of heavy SILAC-labelled proteins in HeLa S3 cells treated with 2′–5′ OA or OH-of 2′–5′ OA (n = 4). Diagonal lines indicate a slope of 1, including an offset of ± log 2 0.5 in case of dashed lines. (C) As (B) in HeLa cells with low RNase L levels. (D, E) MEFs were treated with siRNA for ABCF1 or non-targeting control for 48 h and treated with the indicated stimuli. (D) Abundance of ABCF1 normalized to GAPDH transcripts in relation to mock siCTRL. Graph shows mean (± sd, n = 2) of one representative experiment of two. (E) MEF cells were treated with different stimuli to induce type-I interferon production. After 24 h the supernatants were assayed for presence of typ-I interferon using a bioassay on ISRE-Luc reporter containing L292 cell line. Gaph shows mean (± sd, n = 3) of one representative experiment of three.

Article Snippet: Antibodies against the following proteins were used: ABCF1 (Aviva Systems biology; ARP43631_P050), ABCF3 (Sigma; HPA036332), RNASE L (Abcam; ab13825), HA tag (HRP coupled, Sigma; H6533), β-actin (HRP coupled, Santa Cruz; sc-47778) and HRP coupled secondary antibodies against rabbit IgG (Cell Signaling Technology; 7074) and against murine IgG (Columbia Biosciences; HRP-112).

Techniques: Multiplex sample analysis, Transformation Assay, Control, Bioassay

Journal: bioRxiv

Article Title: RNase L activating 2′–5′ oligoadenylates bind ABCF1, -3 and Decr-1

doi: 10.1101/2023.03.21.532770

Figure Lengend Snippet: (A) Comparison of fraction of 2′–5′ OA bound with or without the 5′ triphosphate. Calf-intestinal phosphatase treatment removes the 5′ triphosphate and reduces mDecr1-2′–5′ OA bound complex formation. (B) 1.35 Å crystal structure of mouse Decr1 bound to 2′–5′ OA. Decr1 is a homo-tetrameric metabolic auxiliary enzyme that catalyzes the reduction of trans-unsaturated fatty acids in the mitochondria. We see electron density for one molecule of 2′–5′ OA with three clearly defined bases bound to each monomer of Decr1. (C) 2Fo-Fc map of 2′–5′ OA bound to one monomer of Decr1 contoured to 1 σ. (D) 2′–5′ OA bound to mouse Decr1. 2′–5′ OA contacts charged residues in each Decr1 monomer with specific contacts to the 2′ phosphate and first base. Binding pocket and active site residues in mDecr1 and hDecr1 are highly conserved (compare to E). (E) NADPH bound to human Decr1 (PDB: 1W6U) with 2′–5′ OA modeled and overlayed. 2′–5′ OA binds in an extended binding site compared to NADPH, which has key contacts deeper in the binding pocket. N92 in mDecr1 is permissive to 2′–5′ OA binding, while K92 in hDecr1 allows NADPH binding but sterically clashes with modelled 2′–5′ OA binding. (F) Comparison of mouse Decr1, human Decr1, or human mutant K92N and 2′–5′ OA complex formation. WT human Decr1 cannot form a complex with 2′–5′ OA, while the K92N mutation in the human Decr1 background is permissive to 2′–5′ OA binding and complex formation. (G) NIH-3T3 cells were transduced with lentiviral vectors expressing Cas9 and gRNA targeting Abcf1, Abcf3, Decr1, Mavs or non-targeting control (Neg). Targeted cell populations after puromycin selection were infected with viruses encoding fluorescent reporter genes. The graphs show mean fluorescent intensity (± sd, n = 4) normalized to cell confluence for timepoints 27 hpi (VSV-eGFP MOI 0.1), 48 hpi (RVFV-Katushka MOI 0.1), 60 hpi (YFV-Venus MOI 0.5), 72 hpi (HSV-1-eGFP MOI 1) or 96 hpi (VACV-eGFP MOI 0.01).

Article Snippet: Antibodies against the following proteins were used: ABCF1 (Aviva Systems biology; ARP43631_P050), ABCF3 (Sigma; HPA036332), RNASE L (Abcam; ab13825), HA tag (HRP coupled, Sigma; H6533), β-actin (HRP coupled, Santa Cruz; sc-47778) and HRP coupled secondary antibodies against rabbit IgG (Cell Signaling Technology; 7074) and against murine IgG (Columbia Biosciences; HRP-112).

Techniques: Comparison, Binding Assay, Mutagenesis, Transduction, Expressing, Control, Selection, Infection

Journal: bioRxiv

Article Title: Phagocytosis in macrophages is regulated by the ATP-binding cassette family gene ABCF1

doi: 10.1101/2023.09.05.556419

Figure Lengend Snippet: A) In bone marrow-derived macrophages (BMDMs), ABCF1 was manipulated through either overexpression (Over Exp.) or treatment with specific siRNA. These interventions were carried out both in the presence and absence of lipopolysaccharide (LPS) stimulation (at a concentration of 100 ng/ml for 24 hours). The resulting heat map illustrates the fold change in various cytokines and chemokines detected in the cell culture supernatants. B) Similarly, in BMDMs, ABCF1 was modulated using Abcf1-specific siRNA, and these cells were exposed to LPS treatment. The heat map presented here showcases the fold change in various Mitogen-Activated Protein Kinase (MAPK) and Interferon-Stimulated Gene (ISG)-specific phosphoproteins and transcription factors detected in whole-cell lysates (WCL).

Article Snippet: Specifically, ABCF1 gene knockdown was accomplished using Mouse Abcf1 siRNA from Santa Cruz Biotechnology (catalog number sc-140760), resulting in a consistent reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Derivative Assay, Over Expression, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: Phagocytosis in macrophages is regulated by the ATP-binding cassette family gene ABCF1

doi: 10.1101/2023.09.05.556419

Figure Lengend Snippet: A) The absorbance value associated with phagocytosis was assessed in bone marrow-derived macrophages (BMDMs) that were subjected to ABCF1-specific siRNA treatment. These BMDMs were incubated with fluorescently labeled IgG-coated latex beads for a duration of 2 hours, both in the presence and absence of lipopolysaccharide (LPS). Additionally, ABCF1-specific siRNA-treated BMDMs were incubated with fluorescently labeled E. coli for 2 hours. The results provide insights into the impact of ABCF1 modulation on phagocytosis; B ) The levels of CD16 and CD64 were analyzed using flow cytometry in treated BMDMs. The bar graph depicts the change in mean fluorescence intensity (MFI), and error bars indicate the standard deviation. C ) The levels of CD32 were analyzed using flow cytometry in treated BMDMs. The bar graph depicts the change in mean fluorescence intensity (MFI), and error bars indicate the standard deviation. This analysis sheds light on how ABCF1 manipulation influences CD32 expression; C ) In BMDMs, ABCF1 was either overexpressed or not, and these cells were exposed to LPS. Whole-cell lysates (WCL) were immunoprecipitated with an anti-CD32 antibody. The immunoprecipitates were subsequently subjected to immunoblotting using anti-CD32 and anti-phosphotyrosine antibodies. This experimental approach allows the investigation of ABCF1’s role in CD32-associated signaling events; D ) The heat map represents the fold change in phosphorylation levels of Src Family Kinases (SFKs) detected in whole-cell lysates (WCL) from ABCF1-specific siRNA-treated and LPS-stimulated BMDMs. This analysis provides insights into how ABCF1 modulation affects SFK phosphorylation levels in this context.

Article Snippet: Specifically, ABCF1 gene knockdown was accomplished using Mouse Abcf1 siRNA from Santa Cruz Biotechnology (catalog number sc-140760), resulting in a consistent reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Derivative Assay, Incubation, Labeling, Flow Cytometry, Fluorescence, Standard Deviation, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Phagocytosis in macrophages is regulated by the ATP-binding cassette family gene ABCF1

doi: 10.1101/2023.09.05.556419

Figure Lengend Snippet: ABCF1 plays a crucial role in orchestrating the intricate process of phagocytosis, particularly in the engulfment of E. coli particles mediated by Fcγ receptors (FcγRs). The sequence of events involving ABCF1 in this process unfolds as follows: 1 ) ABCF1 is essential for the phosphorylation of Immunoreceptor Tyrosine-Based Activation Motifs (ITAMs) by Src Family Kinases (SFKs) such as SRC, LYN, or FGR. This initial phosphorylation event is the trigger for FcγR-mediated phagocytosis of E. coli particles; 2 ) Following ITAM phosphorylation, ABCF1 takes on the role of targeting Spleen Tyrosine Kinase (SYK) for K63-linked polyubiquitination. This ubiquitination event is crucial for subsequent signaling events; 3 ) SYK, once polyubiquitinated, undergoes phosphorylation mediated by SFKs, such as FYN and other members of the SFK family. These phosphorylation events initiate downstream signaling pathways; 4 ) Phosphorylated SYK sets off a cascade of events, including the phosphorylation of Phospholipase C Gamma 2 (PLCγ2), FYN, and other SFKs. These signaling molecules play critical roles in actin polymerization and the formation of the phagocytic cup, a structural prerequisite for successful phagocytosis; 5 ) The downstream consequences of this signaling cascade include elevated phosphorylation levels of Signal Transducer and Activator of Transcription (STAT) proteins, particularly STAT 2, 3, and 6. These phosphorylated STAT proteins contribute to an increase in the production of anti-inflammatory cytokines. In summary, ABCF1’s involvement in phagocytosis of E. coli particles revolves around its role in initiating and coordinating a series of signaling events that ultimately lead to successful engulfment and the production of anti-inflammatory cytokines.

Article Snippet: Specifically, ABCF1 gene knockdown was accomplished using Mouse Abcf1 siRNA from Santa Cruz Biotechnology (catalog number sc-140760), resulting in a consistent reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Sequencing, Phospho-proteomics, Activation Assay, Ubiquitin Proteomics, Protein-Protein interactions

Journal: Experimental & Translational Stroke Medicine

Article Title: ATP-binding cassette transporters in immortalised human brain microvascular endothelial cells in normal and hypoxic conditions

doi: 10.1186/2040-7378-4-9

Figure Lengend Snippet: Expression of ABC transporters in the immortalised human brain microvascular endothelial cell line hCMEC/D3

Article Snippet: ABCF1 , Hs00153703_m1 , ●● , 15.21 , (0.44) , 2.44 , (1.80-3.31).

Techniques: Expressing

Journal: Experimental & Translational Stroke Medicine

Article Title: ATP-binding cassette transporters in immortalised human brain microvascular endothelial cells in normal and hypoxic conditions

doi: 10.1186/2040-7378-4-9

Figure Lengend Snippet: Expression and regulation of ABC transporters in the immortalised human brain microvascular endothelial cell line hCMEC/D3 after hypoxia and after ischemia/reperfusion

Article Snippet: ABCF1 , Hs00153703_m1 , ●● , 15.21 , (0.44) , 2.44 , (1.80-3.31).

Techniques: Expressing