Journal: NPJ Vaccines

Article Title: Immunization-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection

doi: 10.1038/s41541-024-00856-6

Figure Lengend Snippet: a Experimental schematic for b – e . C57/BL6 mice were immunized subcutaneously in the footpad and/or flank with the indicated antigens and adjuvants. b Cells were stained with CD45, PDPN, CD31 and PD-L1. Cells were gated on CD45-PDPN + CD31- for FRC and CD45-PDPN + CD31+ for LECs. Shown are examples of LEC and FRC antigen-positive cells based on PD-L1 expression (floor, MARCO LEC) and ova-AF488+ from mice 2–3 weeks after immunization with ova conjugated to Alexa-Fluor 488 (AF488) and polyI:C and αCD40. c Quantification of the frequency of LEC, BEC, and FRC in the popliteal lymph node (pLN) that are positive for the indicated antigens administered with polyI:C and αCD40 at indicated time. d Same as ( b ) except for mice were immunized with SARS-CoV-2-RBD-AF488, polyI:C, and αCD40. e Same as in ( c ) except for SARS-CoV-2-RBD and CHIKV-E2 with polyI:C and αCD40. CHIKV-E2 was repeated for 9–14 days post-vaccine (~2 weeks). Statistical analysis was done using an unpaired t -test where the p -value between naïve and indicated antigen is <0.0001. In each experiment, at least n = 2–3 mice per group were evaluated and the experiment was repeated n = 2–5 times for c – e . Shown is the representative data from one of the experiments. Error bars are mean ± standard error of the mean. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Ova (10 μg) was purchased from Sigma-Aldrich (Cat No. A5503) and Chikungunya virus envelope 2 protein (8 μg) (CHIKV-E2, strain SL-CK1) was purchased from Sino Biological (Cat. No. 40440-V08B).

Techniques: Staining, Expressing

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: ( a ) Structure of the binding interface between the UFD of S. pombe Uba1 (interacting residues colored pink) and the alpha-1 helix (aa 1–18, colored by amino acid property) of the E2 Ubc15 (cyan), as revealed by the x-ray structure of the S. pombe Uba1/Ubc15 complex (PDB: 5KNL) . ( b ) Amino acid sequences of the SAH-Ubc15 E7R staple-scanning library, which was generated by inserting all-hydrocarbon i, i+4 or i, i+7 staples sequentially along the length of the Ubc15 E7R peptide. X, S5-pentenyl alanine; 8, R5-octenyl alanine; B, norleucine (replacement for methionine, whose sulfur residue decreases the efficiency of ruthenium-catalyzed olefin metathesis). ( c ) Helical wheel depiction of SAH-Ubc15 E7R peptides and their staple positions (helical residues span from aa 5–18). ( d ) Inhibition of UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2 by the indicated SAH-UBC15 E7R stapled peptides. Thioester transfer activity was monitored by the shift in E2 molecular weight from 17 kDa (free UBE2D2) to 25 kDa (UBE2D2~ubiquitin conjugate), as detected by gel electrophoresis and silver stain. Vehicle lane contained 1.5% DMSO and control lane lacked UBE1 protein. The bar graph represents mean inhibition relative to vehicle control ± s.e.m., as quantified and averaged across three independent biological replicates. Uncropped gel for panel d is available online. Source data for the thioester transfer assay plot is available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Binding Assay, Generated, Residue, Inhibition, Ubiquitin Proteomics, Activity Assay, Molecular Weight, Nucleic Acid Electrophoresis, Silver Staining, Control

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: ( a ) Amino acid sequences of stapled peptides corresponding to the N terminus and alpha-1 helix of UBE2D2 (aa 1–16), UBE2G2 (aa 1–14), and UBE2A (aa 1–17), which incorporate the optimal staple position identified for SAH-Ubc15–11. X, S5-pentenyl alanine; 8, R5-octenyl alanine; B, norleucine. ( b ) Comparative dose-responsive inhibition of UBE1 by the indicated SAH-E2 h1 peptides, as monitored by thioester transfer assay, gel electrophoresis, and silver stain. ( c ) Quantitation of the data in b by ImageJ analysis. Data are mean percent inhibition ± s.e.m. for three independent biological replicates. ( d ) Circular dichroism spectra of UBE2A (BSTPARRRLBRDFKRLQ, where B is norleucine) and SAH-UBE2A, demonstrating induction of α-helicity by insertion of the i, i+7 staple. ( e ) Inhibition of UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2 by SAH-UBE2A but not the corresponding unmodified template peptide, UBE2A. Data are mean inhibition ± s.e.m. for three independent biological replicates. ( f ) Comparative protease resistance of SAH-UBE2A and UBE2A upon treatment with proteinase K, as measured by HPLC analysis. SAH-UBE2A displays a 23-fold longer half-life than UBE2A. Data are mean ± s.d. for experiments performed in triplicate. ( g ) SAH-UBE2A but not UBE2A was significantly protected from deuterium exchange upon incubation with UBE1. The relative difference in deuterium uptake after 10 sec of labeling is shown. The difference in uptake was calculated from the mean deuterium level for UBE2A or SAH-UBE2A in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. ( h ) C-terminally biotinylated SAH-UBE2A directly bound to recombinant human UBE1 (MW 118 kDa), as demonstrated by streptavidin capture, gel electrophoresis, and silver stain. ( i ) C-terminally FITCylated SAH-UBE2A bound to UBE1 with a K d of 384 ± 43 nM, as assessed by fluorescence polarization assay. Error bars are s.d. for assays conducted in technical triplicate and performed three times with independent preparations of peptide and protein (all 9 points for each dosing level are plotted). Uncropped gels for panels b , e , and h are available online. Source data for thioester transfer assays, CD spectra, protease resistance testing, HXMS analyses, and FP plot are available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Inhibition, Nucleic Acid Electrophoresis, Silver Staining, Quantitation Assay, Circular Dichroism, Ubiquitin Proteomics, Incubation, Labeling, Recombinant, Fluorescence

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: (a) Sequence compositions of the unmodified UBE2A template peptide and stapled constructs SAH-UBE2A, SAH-UBE2A R7E , and SAH-UBE2A R7A/L9A/B10A . (b) Comparative effects of UBE2A, SAH-UBE2A, SAH-UBE2A R7E , and SAH-UBE2A R7A/L9A/B10A on UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2. Whereas SAH-UBE2A (10 μM) completely inhibits UBE1 activity, triple mutagenesis abrogates the inhibitory effect and single R7E mutagenesis has an intermediate effect. (c) Progressive loss of UBE1 protection from deuterium exchange into SAH-UBE2A upon single R7E and triple R7A/L9A/B10A mutagenesis. The relative difference in deuterium uptake after 10 sec of labeling is shown. The difference in uptake was calculated from the mean deuterium level for each SAH-UBE2A peptide in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. (d) Difference in deuterium uptake plots demonstrate the relative deuterium incorporation of SAH-UBE2A/UBE1, SAH-UBE2A R7E /UBE1, or SAH-UBE2A R7A/L9A/B10A /UBE1 minus the relative deuterium incorporation of UBE1 at 10 min of deuterium labeling. The shaded gray region indicates differences in deuteration that are below the meaningful differences threshold. Consistent with results of both the thioester transfer assay (b) and peptide deuterium exchange (c), and the predicted SAH-UBE2A/UBE1 binding mode, triple mutagenesis essentially abrogates the effect of SAH-UBE2A on UBE1 conformation, with single R7E mutagenesis having an intermediate influence. Data are representative of three independent replicates for SAH-UBE2A, and two independent replicates for SAH-UBE2A R7E and SAH-UBE2A R7A/L9A/B10A . Each peptide, from N- to C-terminus, was given a peptide number to simplify the presentation. The peptide list and residue identity of each peptide can be found in . Uncropped gel for panel b is available online. Source data for the HXMS analyses are available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Sequencing, Construct, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Labeling, Binding Assay, Residue

Journal: Infectious agents and cancer

Article Title: Silencing of UBE2D1 inhibited cell migration in gastric cancer, decreasing ubiquitination of SMAD4.

doi: 10.1186/s13027-021-00402-2

Figure Lengend Snippet: Fig. 1 UBE2D1 was elevated in GC tissues. A The mRNA level of UBE2D1 was analyzed from TCGA-STAD data. B The mRNA level of UBE2D1 was detected in 25 pairs of tumors and paracancerous samples. ***, P < 0.001. C The protein level of UBE2D1 in tissue array (32 pairs) was determined by IHC staining. D Impact of UBE2D1 expression on OS in GC patients in TCGA

Article Snippet: Primary antibodies were listed as following: UBE2D1 (1:800, Proteintech, Rosemont, IL, USA), SMAD4 (1:1000, Abcam, St. Louis, MO, USA), MMP2 (1:1000, Abcam), MMP9 (1:800, Abcam), and β-actin (1:2000, Abcam).

Techniques: Immunohistochemistry, Expressing

Journal: Infectious agents and cancer

Article Title: Silencing of UBE2D1 inhibited cell migration in gastric cancer, decreasing ubiquitination of SMAD4.

doi: 10.1186/s13027-021-00402-2

Figure Lengend Snippet: Fig. 2 Expression level of UBE2D1 was measured by real-time PCR western blot assay. A UBE2D1 expression level in different cell lines, including GSE-1, AGS, BGC-823, MGC-803, MKN45, and SGC-7901. B Knock-down efficiency was evaluated after transduction with shUBE2D1 lentiviruses in AGS and MKN45 cells. ***, P < 0.001 versus shNC. C Overexpression efficiency was evaluated after transduction with UBE2D1 overexpression lentivirus in MGC-803 cells. ***, P < 0.001 versus vector

Article Snippet: Primary antibodies were listed as following: UBE2D1 (1:800, Proteintech, Rosemont, IL, USA), SMAD4 (1:1000, Abcam, St. Louis, MO, USA), MMP2 (1:1000, Abcam), MMP9 (1:800, Abcam), and β-actin (1:2000, Abcam).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Transduction, Over Expression, Plasmid Preparation

Journal: Infectious agents and cancer

Article Title: Silencing of UBE2D1 inhibited cell migration in gastric cancer, decreasing ubiquitination of SMAD4.

doi: 10.1186/s13027-021-00402-2

Figure Lengend Snippet: Fig. 4 Overexpression of UBE2D1 promoted cell migration in vitro. A–C Transwell (A and B) and wound healing (C) assays were performed to analyze cell migration after transduction with UBE2D1 overexpression lentivirus in MGC-803 cells. ***, P < 0.001 versus vector. D A western blot assay was performed to measure the protein levels of UBE2D1, MMP2, and MMP9

Article Snippet: Primary antibodies were listed as following: UBE2D1 (1:800, Proteintech, Rosemont, IL, USA), SMAD4 (1:1000, Abcam, St. Louis, MO, USA), MMP2 (1:1000, Abcam), MMP9 (1:800, Abcam), and β-actin (1:2000, Abcam).

Techniques: Over Expression, Migration, In Vitro, Transduction, Plasmid Preparation, Western Blot

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: SARS-COV-2 N protein interacts with UBC9. ( A ) pCDNA3.1 (3XFlag Tag) with N protein-coding genes and pEGFP-N1 (GFP Tag) with UBC9-coding genes were co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. ( B ) pDsRED-mono-N1 (RFP Tag) with SARS-COV-2 N protein-coding genes and pEGFP-N1 (GFP Tag) with UBC9 or UBC9 C93A coding genes were expressed or co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cells were stained with DAPI before confocal microscopy imaging. Endogenous UBC9 in HEK293T cells was also detected by FITC-conjugated Goat Anti-Mouse IgG(H + L), which recognizes anti-UBC9 mouse antibodies. Scale bars: 10 μm. ( C ) pET28a (HIS 6 tag, Kan + ) with SARS-COV-2 N protein coding genes and pGEX-6P-1 (GST tag, Amp + ) with UBC9 or UBC9 C93A coding genes were co-expressed in BL21 (DE3) E. coli . cell lysates were subjected to Co-IP after IPTG (1 mM) induction for 6 h. ( D ) pCDNA3.1 (3XFlag Tag) with SARS-COV-2 N protein-coding genes was over-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Transfection, Co-Immunoprecipitation Assay, Staining, Confocal Microscopy, Imaging

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: Human beta coronavirus N protein interacts with UBC9. ( A ) pET28a-N (HIS 6 tag, Kan + ) containing five human beta coronavirus N protein-coding genes and pGEX-UBC9 (GST tag, Amp + ) harboring the UBC9 coding gene were co-expressed in BL21 (DE3) E. coli Cell lysates were prepared and subjected to Co-IP following IPTG induction (1 mM) for 6 h. ( B ) pEGFP-N1 (GFP Tag) with three human beta coronavirus N protein-coding genes and pCDNA3.1 (3XFlag Tag) with the UBC9 coding gene were co-expressed in HEK293T cells. After a 24- h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Co-Immunoprecipitation Assay, Transfection

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: SARS-COV-2 N protein enhanced the molecular interaction between UBC9 and MAVS. ( A ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N protein or UBC9 coding genes and pCDNA3.1 (3XFlag Tag) with MAVS coding gene were co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. ( B ) Relative Ubc9 band intensity before or after IP: Flag in ( A ) were calculated by using ImageJ in triplet replicates. ns: no significant difference, *** p < 0.001. ( C ) Schematic diagram of wild-type SARS-CoV-2 N protein and truncated mutants. ( D ) pEGFP-N1 (GFP Tag) with UBC9 coding genes and pCDNA3.1 (3XFlag Tag) with SARS-COV-2 N protein or truncation coding gene were co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: The expression of UBC9 in HEK293T, Vero, Vero E6, Huh7, and HRT18 cells was detected by Western blotting.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Expressing, Western Blot

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: The interaction between the SARS-CoV-2 N protein and UBC9 inhibits MAVS ubiquitination by enhancing its SUMOylation. ( A , B ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA, or K63-His6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 highly expressed) or Vero E6 cells (endogenous UBC9 underexpressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: The interaction of N protein and UBC9 plays a crucial role in regulating the SUMOylation and ubiquitination modifications of MAVS. ( A ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA or SUMO3 KR -HA, K63-His 6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 was highly expressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. ( B ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA, UBC9 or UBC9 C93A , K63-His 6 coding genes were co-expressed in Vero E6 cells (endogenous UBC9 underexpressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. ( C ) pEGFP-N1 (GFP Tag) with SARS-COV-2 N, N 44–419 or N 174–419 coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA, and K63-His 6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 highly expressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation

doi: 10.3390/v15122304

Figure Lengend Snippet: UBC9 plays a critical role in the process of impaired IFN I response caused by the in-teraction between the N protein and MAVS during virus infection. ( A , B ) pCDNA3.1 with SARS-CoV-2 N protein-coding genes was gradually increased in HEK293T cells or VERO E6 cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Western blotting. ( C , D ) The relative band intensity of Western blotting results (a relative band quantification of phosphorylation protein to total protein) in ( A , B ) were calculated by using ImageJ in triplet replicates. ns: no significant difference, p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Virus, Infection, Transfection, Western Blot, Phospho-proteomics

Journal: Veterinary Microbiology

Article Title: Cellular poly(C) binding protein 2 interacts with porcine epidemic diarrhea virus papain-like protease 1 and supports viral replication

doi: 10.1016/j.vetmic.2020.108793

Figure Lengend Snippet: Primers used for genes amplication.

Article Snippet: Rabbit polyclonal PCBP2 antibody (Proteintech, Chicago, IL) was used to detect endogenous PCBP2 protein.

Techniques: Quantitative RT-PCR, Amplification

Journal: Veterinary Microbiology

Article Title: Cellular poly(C) binding protein 2 interacts with porcine epidemic diarrhea virus papain-like protease 1 and supports viral replication

doi: 10.1016/j.vetmic.2020.108793

Figure Lengend Snippet: Identification of proteins that interact with PLP1 . HEK293 T cells were seeded in a six-well plate and cotransfected with pCMV-PLP1-HA, pECMV-3×FLAG-PCBP1, pECMV-3×FLAG-PCBP2 or empty vector. At 24 hpt, cells were lysed and immunoprecipitated with mouse anti-FLAG antibody. Immunoprecipitated samples were analyzed by western blotting.

Article Snippet: Rabbit polyclonal PCBP2 antibody (Proteintech, Chicago, IL) was used to detect endogenous PCBP2 protein.

Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Veterinary Microbiology

Article Title: Cellular poly(C) binding protein 2 interacts with porcine epidemic diarrhea virus papain-like protease 1 and supports viral replication

doi: 10.1016/j.vetmic.2020.108793

Figure Lengend Snippet: Co-localization of PCBP2 with PLP1 . (A) Vero E6 cells were cotransfected with pCMV-PLP1-HA and pECMV-3×FLAG-PCBP2 or empty pCMV-HA vector. The cells were assessed using an indirect immunofluorescence assay with anti-HA and anti-FLAG antibodies. (B) Co-localization of PLP1 and endogenous PCBP2 in Vero E6 cells. The cells were transfected with pCMV-PLPL-HA or empty pCMV-HA vector and then subjected to the IFA with anti-HA and anti-PCBP2 antibodies.

Article Snippet: Rabbit polyclonal PCBP2 antibody (Proteintech, Chicago, IL) was used to detect endogenous PCBP2 protein.

Techniques: Plasmid Preparation, Immunofluorescence, Transfection

Journal: Veterinary Microbiology

Article Title: Cellular poly(C) binding protein 2 interacts with porcine epidemic diarrhea virus papain-like protease 1 and supports viral replication

doi: 10.1016/j.vetmic.2020.108793

Figure Lengend Snippet: PLP1 up-regulates PCBP2 expression . Vero 6 cells were transfected with different concentrations of pCMV-PLP1-HA or empty pCMV-HA vector and western blotting was performed using anti-HA, anti-PCBP2 and anti-tubulin antibodies.

Article Snippet: Rabbit polyclonal PCBP2 antibody (Proteintech, Chicago, IL) was used to detect endogenous PCBP2 protein.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: Veterinary Microbiology

Article Title: Cellular poly(C) binding protein 2 interacts with porcine epidemic diarrhea virus papain-like protease 1 and supports viral replication

doi: 10.1016/j.vetmic.2020.108793

Figure Lengend Snippet: PCBP2 affects PEDV infection . IPEC-J2 cells were transfected with three different PCBP2-specific siRNAs. After 24 h, PCBP2 protein expression was assessed by western blotting with anti-PCBP2 and anti-tubulin antibodies. (B to C) IPEC-J2 cells were transfected with siRNA1, then 24 h later infected with PEDV for 24 h and 48 h. PCBP2 mRNA levels and PEDV RNA levels were determined by quantitative RT-PCR. (D) IPEC-J2 cells were transfected with pECMV-3×FLAG-PCBP2 or empty vector. At 24 hpt, the cells were infected with PEDV at a MOI of 0.1. PEDV viral loads were determined at 24, 48 and 60 hpi by quantitative RT-PCR.

Article Snippet: Rabbit polyclonal PCBP2 antibody (Proteintech, Chicago, IL) was used to detect endogenous PCBP2 protein.

Techniques: Infection, Transfection, Expressing, Western Blot, Quantitative RT-PCR, Plasmid Preparation