Journal: Oncology reports

Article Title: Decreased concentrations of retinol-binding protein 4 in sera of epithelial ovarian cancer patients: a potential biomarker identified by proteomics.

doi: 10.3892/or.2011.1513

Figure Lengend Snippet: Figure 3. Verification of altered RBP4 concentration. (A) Absolute RBP4 con centrations in serum samples were determined by ELISA. Each measurement represents average value from 3 replicates. Individual serum RBP4 concen trations can be found in Tables I and II. (B) Western blot analysis of RBP4 concentration in individual serum samples was performed. To enable relative comparison between healthy controls and patients five control and five patient samples (10 µg protein each) were run on each gel and processed simultane ously. Membranes were re-probed with anti-human IgG (HC) as an internal standard.

Article Snippet: The quantitative determination of human retinolbinding protein 4 (RBP4), α-1-antitrypsin and apolipoprotein A4 concentrations in patient and control sera was performed in triplicates using the Quantikine Human RBP4 immunoassay (R&D Systems, MN, USA), human apolipoprotein AIV ELISA Kit (Millipore, MA, USA) and α-1-antitrypsin Clearance ELISA (Immuno Diagnostik AG, Germany) according to the manufacturer's instructions using ELISA Reader Sunrise (Tecan, Austria).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison, Control

Journal: Oncology reports

Article Title: Decreased concentrations of retinol-binding protein 4 in sera of epithelial ovarian cancer patients: a potential biomarker identified by proteomics.

doi: 10.3892/or.2011.1513

Figure Lengend Snippet: Figure 4. Serum retinol levels in EOC patients and age-matched healthy women. Levels of serum vitamin A (retinol) regulate RBP4 secrection by liver. Decreased retinol levels could affect RBP4 levels in our study. We therefore determined serum retinol levels in all 10 patients and controls by HPLC. Mean values of serum retinol concentrations are shown.

Article Snippet: The quantitative determination of human retinolbinding protein 4 (RBP4), α-1-antitrypsin and apolipoprotein A4 concentrations in patient and control sera was performed in triplicates using the Quantikine Human RBP4 immunoassay (R&D Systems, MN, USA), human apolipoprotein AIV ELISA Kit (Millipore, MA, USA) and α-1-antitrypsin Clearance ELISA (Immuno Diagnostik AG, Germany) according to the manufacturer's instructions using ELISA Reader Sunrise (Tecan, Austria).

Techniques:

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: Immunoblotting analysis of RBP4 protein levels and PCV2 capsid protein (Cap) expression in 3D4/21 cells and PK-15 cells infected with PCV2 (MOI = 0.2, the same dose below) at the indicated periods ( a ) or infected with PCV2 with increased dose for 36 h ( b ). Quantitative real-time PCR (qPCR) analysis of RBP4 mRNA expression in 3D4/21 cells and PK-15 cells infected with PCV2 at the indicated periods ( c ) or infected with PCV2 with increased dose for 36 h ( d ). e Immunoblotting analysis of RBP4 protein expression in 3D4/21 cells and PK-15 cells left untreated or infected with PCV2 for 36 h following treatment with cycloheximide (CHX, 50 μM) for the indicated periods. Densitometric quantitation of RBP4 was normalized relative to the levels at 0 h conditions. f Immunoblotting analysis of RBP4, phosphorylated (p-) and total p38, p-JNK and JNK, p-p65 and p65, and p-ERK1/2 and ERK1/2 in whole lysates of in 3D4/21 cells or PK-15 cells pretreated with DMSO or p38 inhibitor SB203580 (SB, 10 μM), JNK inhibitor SP600125 (SP, 10 μM), NF-κB inhibitor BAY11 (10 μM), or ERK inhibitor U0126 (10 μM) for 3 h followed by PCV2 infection for 36 h. g Immunoblotting analysis of RBP4, phosphorylated (p-) and total eIF4E, p-p38 and p38, and p-ERK1/2 and ERK1/2 in whole lysates of 3D4/21 cells and PK-15 cells left untreated or infected with PCV2 for the indicated periods. h Immunoblotting analysis of RBP4, p-eIF4E, and total eIF4E in whole-cell lysates of 3D4/21 cells and PK-15 cells left untreated or pretreated with DMSO, U0126 (10 μM), or SB (10 μM) for 3 h followed by PCV2 infection for the indicated periods. Data are representative of three independent experiments ( a , b , e – h ) or pooled from three independent experiments ( c , d , mean ± SD).

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitation Assay

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: a Immunoblotting analysis of RBP4 and PCV2 ORF protein expression in whole-cell lysates of 3D4/21 cells and PK-15 cells transfected with empty vector (EV) or PCV2 ORF (ORF1–ORF5)-expressing plasmids for 24 h. b Immunoblotting analysis of RBP4 protein levels in whole-cell lysates of 3D4/21 cells and PK-15 cells transfected with EV or PCV2 ORF1-expressing plasmid with increased dose for 24 h. c qPCR analysis of RBP4 mRNA levels in 3D4/21 cells and PK-15 cells transfected with EV or PCV2 ORF1 plasmid for the indicated periods. d Immunoblotting analysis of RBP4, p-eIF4E, and total eIF4E in whole-cell lysates of 3D4/21 cells and PK-15 cells transfected with EV or PCV2 ORF1-5 plasmids for 24 h. e Immunoblotting analysis of RBP4, p-eIF4E and total eIF4E, p-p38 and total p38, and p-ERK1/2 and total ERK1/2 in whole-cell lysates of 3D4/21 cells and PK-15 cells transfected with EV or PCV2 ORF1 plasmid for the indicated periods. Data are representative of three independent experiments ( a , b , d , and e ) or pooled from three independent experiments ( c , mean ± SD).

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: Immunoblotting analysis of PCV2 Cap protein levels ( a ) or TCID 50 assay of viral titers ( b ) in 3D4/21 cells (left panel) or PK-15 (right panel) cells transfected with empty vector or Flag-RBP4 plasmid for 12 h followed by PCV2 infection for the indicated periods or 48 h. Immunoblotting analysis of PCV2 Cap protein levels ( c ) or TCID 50 assay of viral titers ( d ) in wild-type (WT) and RBP4-deficient (RBP4-KO) 3D4/21 cells (left panel), WT and RBP4-KO PK-15 cells (right panel) infected with PCV2 for indicated periods or 48 h. Immunoblotting analysis of PCV2 Cap protein levels ( e ) or TCID 50 assay of viral titers ( f ) in RBP4-KO 3D4/21 cells (left panel) and RBP4-KO PK-15 cells (right panel) infected with PCV2 for the indicated periods or 48 h. After 6 h of PCV2 infection, cells were treated with or without recombinant porcine RBP4 (rpRBP4, 30 μg/mL). g Immunoblotting analysis of PCV2 Cap protein levels (left panel) or TCID 50 assay of viral titers (right panel) in primary PAMs infected with PCV2 for the indicated periods or 48 h. After 6 h of PCV2 infection, the cells were treated with or without recombinant porcine RBP4 (rpRBP4, 30 μg/mL). h Immunoblotting analysis of PCV2 Cap protein levels (left panel) or TCID 50 assay of viral titers (right panel) in WT or RBP4-KO BMDMs infected with PCV2 for the indicated periods or 48 h. i Hematoxylin and eosin (H&E) staining of lung (left) and liver (right) sections from mice infected with PCV2 (5 × 10 5 pfu/mouse) for 7 days. Scale bar, 20 μm. Original magnification, ×40. j Histological scores of lung and liver from mice infected by PCV2 as in ( i ). Each symbol represents an individual mouse ( n = 6/group). k Viral titers in the lung (left) and liver (right) from WT and RBP4-KO mice ( n = 6/group) infected with PCV2 in ( i ). Data are pooled from three independent experiments ( b , d , f and g , h right, j , k , mean ± SD) or representative of three independent experiments ( a , c , e and g , h left, i ). * p < 0.05, ** p < 0.01 (Student’s t test).

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, Transfection, Plasmid Preparation, Infection, Recombinant, Staining

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: a Immunoblotting analysis of Myc-tagged protein expression of PCV2 ORF1 (left), ORF2 (middle), or ORF3 (right) in whole-cell lysates of WT and RBP4-KO 3D4/21 cells transfected with the expression plasmids for the indicated periods, respectively. b Immunoblotting analysis of PCV2 ORF1 protein levels in whole-cell lysates of WT and RBP4-KO 3D4/21 cells transfected with PCV2 ORF1 expression plasmid for 24 h following treatment with CHX alone (left) (50 μM), CHX together with MG-132 (middle) (30 μM), or CHX together with CQ (right) (20 μM) for the indicated times. Densitometric quantitation of PCV2 ORF1 was normalized relative to the levels of 0 h conditions ( b , lower). c Immunoblotting analysis of PCV2 ORF1 protein expression in whole-cell lysates of RBP4-KO 3D4/21 cells transfected with the PCV2 ORF1 expression plasmid for 24 h and then left untreated or stimulated with rpRBP4 protein (30 μg/mL) for 1 h following treatment with CHX alone, CHX together with MG132, or CHX together with CQ for the indicated times. Densitometric quantitation of PCV2 ORF1 was normalized relative to the levels at 0 h conditions ( c , lower). Immunoblotting analysis of PCV2 ORF1 protein expression in 3D4/21 cells transfected with PCV2 ORF1 expression plasmid for 6 h following treatment with CQ (20 μM) ( d ) or rapamycin (1 μM) ( e ) for the indicated periods. f Immunoblotting analysis of LC3 expression in RBP4-KO 3D4/21 cells stimulated with rpRBP4 with increased dose for 6 h (upper) or stimulated with rpRBP4 protein (30 μg/mL) for indicated periods (lower). Data are representative of three ( a , d , e ) or two ( b , c , f ) independent experiments.

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitation Assay

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: a Immunoprecipitation analysis of WT and RBP4-KO 3D4/21 cells transfected with PCV2 ORF1 and His-Ubiquitin (His-Ub) or His-Ub at K6 (His-Ub-K6), K11 (His-Ub-K11), K27 (His-Ub-K27), K29 (His-Ub-K29), K33 (His-Ub-K33), K48 (His-Ub-K48) or K63 (His-Ub-K63) only for 24 h. b Immunoprecipitation analysis of RBP4-KO 3D4/21 cells expressing PCV2 ORF1 and His-Ub, His-Ub-K48, His-Ub-K63, His-Ub-K48R, or His-Ub-K63R as indicated. Cells were left untreated or stimulated with rpRBP4 protein for 6 h before harvest. c Immunoprecipitation analysis of 3D4/21 cells expressing GFP-TRAF6 together with empty vector (EV), PCV2 ORF1, PCV3 ORF1, or PCV4 ORF1 as indicated. Anti-GFP immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody. Levels of the transfected proteins were analyzed by immunoblotting with anti-Myc and anti-GFP antibodies. d Colocalization of exogenous TRAF6 and PCV2 ORF1 in 3D4/21 cells. Cells were transfected with GFP-TRAF6 and PCV2 ORF1 for 24 h before confocal microscopy. Scale bar, 10 μm. e GST pull-down analysis of the interaction between His-ORF1 and GST, GST-SQSTM1/p62, GST-LC3, or GST-TRAF6 as indicated. Recombinant proteins were pulled down by GST magnetic beads and were analyzed by immunoblotting with anti-His or anti-GST antibodies (upper). Recombinant proteins in the assay were examined by SDS–PAGE and coomassie blue staining (lower). f Schematic drawings of the TRAF6-binding motif Pro-X-Glu-X-X-Ar/Ac. The presence of the putative Pro-X-Glu-X-X-Ar/Ac motifs in ORF1 of PCV2 and other representative circoviruses (PCV3, KX778720.1(Genebank number); PCV4, MK986820.1; PCV1, U49186.1; HuCV2, ON226770.2; GoCV, MT831941.1; DuCV, MN078101.1; and CaCV, JQ821392.1) were analyzed and consistent amino acids are indicated in color. g Immunoprecipitation analysis of the association of PCV2 ORF1 and TRAF6 in 3D4/21 cells transfected with GFP-TRAF6 and wild-type PCV2 ORF1 (WT), or PCV2 ORF1 mutants (P309T, E311A, or P309T/E311A). Anti-Myc immunoprecipitates were analyzed by immunoblotting with anti-GFP or anti-Myc antibody as indicated. Levels of the transfected proteins were analyzed by immunoblotting with anti-GFP or anti-Myc antibody. h Immunoprecipitation analysis of 3D4/21 cells expressing PCV2 ORF1 and His-Ub, His-Ub-K6, His-Ub-K11, His-Ub-K27, His-Ub-K29, His-Ub-K33, His-Ub-K48 or His-Ub-K63 together with or without GFP-TRAF6 as indicated. i Immunoblotting analysis of the indicated proteins in immunoprecipitated samples and whole-cell lysates of RBP4-KO 3D4/21 cells transfected with TRFA6 siRNA or control siRNA (100 nM). Twenty-four hours after transfection, cells were further transfected with PCV2 ORF1 and His-Ub (left) or His-Ub-K63 (right) for 24 h. Cells were then stimulated with rpRBP4 (30 μg/mL) for 6 h before analysis. Data are representative of three ( c – g ) or two ( a , b , h , i ) independent experiments.

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Immunoprecipitation, Transfection, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Western Blot, Confocal Microscopy, Recombinant, Magnetic Beads, SDS Page, Staining, Binding Assay, Control

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: Immunoblotting analysis of PCV2 Cap protein level ( a ) or qPCR analysis of PCV2 DNA copies ( b , left) or TCID50 assay of viral titers ( b , right) in RBP4-KO 3D4/21 cells transfected with empty vector (EV) or Flag-pRBP4 plasmid for 6 h, then cells were treated for 6 h with or without TLR4-specific inhibitor TAK242 (1 μM), followed by infection with PCV2 for the indicated periods or 48 h. Numbers (lower) indicate the grayscale analysis on the protein bands of Cap and RBP4. c Immunoprecipitation analysis of RBP4-KO 3D4/21 cells expressing PCV2 ORF1 and His-Ub-K63 together with or without Flag-RBP4 as indicated for 6 h. Cells were untreated or treated with TAK242 (1 μM) for 18 h before harvest. Data are representative of three independent experiments ( a , c ) or pooled from three independent experiments ( b , mean ± SD). * p < 0.05, ** p < 0.01 (Student’s t test).

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, TCID50 Assay, Transfection, Plasmid Preparation, Infection, Immunoprecipitation, Expressing

Journal: Annals of clinical and translational neurology

Article Title: Proteomics and mathematical modeling of longitudinal CSF differentiates fast versus slow ALS progression.

doi: 10.1002/acn3.51890

Figure Lengend Snippet: Figure 6. Receiver operator characteristic (ROC) curves using targeted measurements of candidate markers of progression by ELISA in the (A) discovery and (B) validation cohorts. Respective area under the curve (AUC) for SERPINA4 (green), RBP4 (red), F12 (blue), and combination (purple) are provided.

Article Snippet: Measurements of human retinol binding protein 4 (RBP4) and kallistatin (SERPINA4) were performed using DuoSet ELISA kits (R&D systems; Minneapolis,per MN) following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery