Journal: Journal of Hematology & Oncology

Article Title: Intracellular delivery of anti-BCR/ABL antibody by PLGA nanoparticles suppresses the oncogenesis of chronic myeloid leukemia cells

doi: 10.1186/s13045-021-01150-x

Figure Lengend Snippet: The role of Trim-Away pathway in Ab@Tf-Cou6-PLGA NPs-mediated BCR/ABL degradation. a Proteasome inhibitor MG132 (100 nM) treatment weakened the BCR/ABL degradation effects of Ab@Tf-Cou6-PLGA NPs in CML cells. b The suppression effect of Ab@Tf-Cou6-PLGA NPs on BCR/ABL was reduced by TRIM21 knockdown. c Immunofluorescence assay was used to analyze the colocalization of BCR/ABL and TRIM21 after Ab@Tf-PLGA NPs treatment. Scale bar, 10 μm. d Co-IP analysis of the interaction of BCR/ABL and TRIM21 with Ab@Tf-Cou6-PLGA NPs treatment. e Co-IP analysis of the interaction of BCR/ABL and TRIM21 without Ab@Tf-Cou6-PLGA NPs treatment

Article Snippet: Co-Immunoprecipitation (Co-IP) experiments were performed with mouse anti-BCR/ABL antibody (Santa, USA), mouse anti-TRIM21 antibody (Santa, USA) and Protein A/G Magnetic beads (MCE, USA).

Techniques: Knockdown, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: Mascot search results of mass spectrometric peptides sequencing of tyrosine phosphorylated proteins following FcγRI cross linking of THP-1 cells (n = 3).

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Sequencing, Control, Ubiquitin Proteomics, Binding Assay, Variant Assay, Activation Assay

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: ( A ) Representative immunoprecipitation of THP-1 cell lysates using anti-pTyr mAb (4G10) followed by Western blotting using selected antibodies showed abundant Tyr phosphorylation of FcγR s, clatherin, HSP70, Cbl, HGS and TRIM21 in cells co-ligated with anti-FcγRI+IgG 1 control mAb validating LC-MS/MS data. Importantly, LILRB4 co-ligation with FcγRI markedly reduced Tyr phosphorylation of all proteins except for HSP70 (n = 3). ( B ) Summary of densitometry of bands from 3 independent experiments showed significant reduction of FcγRs, clatherin, Cbl, HGS and TRIM21 phosphorylation, but not HSP70, in THP-1 cells co-ligated with anti-FcγRI and anti-LILRB4 mAbs, compared to cells co-ligated with anti-FcγRI and negative control mAb (n = 3, **p < 0.01; ***p < 0.001). ( C ) Representative Western blotting of total cell lysates showed that co-ligation of FcγRI with LILRB4 did not alter the total amounts of any of the above proteins when compared to co-ligation of FcγRI+IgG 1 control, ligation of LILRB4 alone or treatment with IgG 1 control alone; the lower panel is the same membrane stripped and re-probed with anti-β actin Ab, confirming comparable protein loading. ( D ) Summary of densitometry analysis of 3 independent experiments showed no significant differences in total FcγRs, clatherin, HSP70, Cbl, HGS and TRIM21 in THP-1 cells within the 4 different treatment groups (n = 3). Full image of the Western blots is shown in . ( E ) Western blotting of cell lysates from FcγRI cross-linked cells showing increased Tyr-phosphorylated Syk that was markedly reduced upon co-ligation with LILRB4, confirming our earlier finding and validating current LC-MS/MS data (n = 1).

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Control, Liquid Chromatography with Mass Spectroscopy, Ligation, Negative Control, Membrane

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: Cross-linking of FcγRI by immune-complexes causes Tyr phosphorylation of the ITAMs of its common γ chain and binding of pSyk transduces activating signals. This simultaneously initiates phosphorylation of clatherin that causes lateral diffusion of receptor-ligand complexes to clathrin-coated pits, membrane invagination and generation of clathrin-coated vesicles, and/or initiates phosphorylation of Cbl that may directly ubiquitinate the receptor. Phosphorylated Cbl triggers phosphorylation of HSP70 that facilitates un-coating of the vesicles, a precondition for vesicles to fuse with early endosomes and release ligands. The released receptors are transported to either the late endosome and/or lysosome for proteosomal and/or lysosomal degradation or are recycled to the cell surface. The immune complexes in the endosome are either directly degraded by Cbl, or delivered to the lysosome by phosphorylated HGS-STAM 1/2 complex for final degradation. During transfer, immune complexes that escape the endosome are recognised by phosphorylated TRIM21 for proteasomal degradation. Co-ligation of FcγRI with LILRB4 may recruit phosphatases such as SHP-1 to its ITIMs that subsequently dephosphorylate (deactivate) the key molecules including clathrin (1), FcγRI and Syk (2), Cbl (3), HGS and STAM 1/2 (4) and TRIM21(5). These effects may reduce cellular activation and/or suppress receptor/ligand endocytosis. *New Tyr phosphorylated and dephosphorylated proteins identified in this study.

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Phospho-proteomics, Binding Assay, Diffusion-based Assay, Membrane, Ligation, Activation Assay

Journal: Journal of Virology

Article Title: Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway

doi: 10.1128/JVI.01684-19

Figure Lengend Snippet: NSs activates the Nrf2 pathway and induces Nrf2-mediated gene expression. (A and B) (Left) RAW 264.7 cells stably expressing the vector, NSs-WT, or NSs-A46 were harvested, and WCEs (A) and the cytoplasmic/nuclear fractions (B) were analyzed by immunoblotting for the indicated proteins. (Right) The relative levels of Nrf2 in RAW 264.7 cells compared to the levels of β-actin for WCEs (A) and p84 for the nucleus (B) were calculated. (C and D) The mRNA levels of the Nrf2 target genes (Hmox1 and Nqo1) (C) and Nrf2 (D) in RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 were measured by qRT-PCR. (E) The Nqo1 mRNA level in RAW 264.7 cells treated with dimethyl sulfoxide (DMSO) and an Nrf2 inhibitor (INH; 10 μM) for 24 h was measured by qRT-PCR.

Article Snippet: The primary antibodies included TRIM21 (clone D1O1D; Cell Signaling), Nrf2 (clone D1Z9C; Cell Signaling), p62 (Cell Signaling), Keap1 (clone D6B12; Cell Signaling), GFP (clone B-2; Santa Cruz), GST (clone B-14; Santa Cruz), β-actin (clone C4; Santa Cruz), Flag (for rabbit Flag, clone F7425 [Sigma]; for mouse Flag, clone F1804 [Sigma]), HA (for rabbit HA, clone PRB-101P [Covance]; for mouse HA, clone 16B12 [BioLegend]), V5 (for rabbit V5, clone A190 [Bethyl]; for mouse V5, Invitrogen), and Myc (for rabbit Myc, clone Poly9063 [BioLegend]; for mouse Myc, clone 9E10 [BioLegend]).

Techniques: Gene Expression, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway

doi: 10.1128/JVI.01684-19

Figure Lengend Snippet: NSs induces CD36 expression via the Nrf2 pathway and increases lipid uptake. (A) The level of Cd36 mRNA in RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 was measured by qRT-PCR. (B) CD36 surface expression by RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 was measured by flow cytometry. The surface expression of FITC fluorescence on cells was determined using FlowJo software (left), and the relative expression levels are provided (right). FACS, fluorescence-activated cell sorting. (C) The level of Cd36 mRNA in RAW 264.7 cells treated with DMSO or an Nrf2 inhibitor (INH, 10 μM) for 24 h was measured by qRT-PCR. (D) RAW 264.7 cells were treated with rabbit IgG-FITC complexed with latex beads at a final dilution of 1:500. (Right) The cells were washed/harvested, and flow cytometry was applied to measure the internalized rabbit IgG-FITC-complexed latex beads. (Left) The percentage of cells that phagocytosed the beads was determined using FlowJo software. (E) The organic phase of RAW 264.7 cells was extracted to measure the amount of free or total (free and esterified) cholesterol. A colorimetric assay was used to measure intracellular cholesterol levels at an absorbance of 570 nm, based on cholesterol standards. (F) RAW 264.7 cells were plated and treated with fluorescently tagged cholesterol at 20 μg/ml for 48 h. The cells were washed, fixed with paraformaldehyde, and applied to a microplate reader with filter sets designed for FITC and GFP. The amount of cholesterol taken up was described at a relative level.

Article Snippet: The primary antibodies included TRIM21 (clone D1O1D; Cell Signaling), Nrf2 (clone D1Z9C; Cell Signaling), p62 (Cell Signaling), Keap1 (clone D6B12; Cell Signaling), GFP (clone B-2; Santa Cruz), GST (clone B-14; Santa Cruz), β-actin (clone C4; Santa Cruz), Flag (for rabbit Flag, clone F7425 [Sigma]; for mouse Flag, clone F1804 [Sigma]), HA (for rabbit HA, clone PRB-101P [Covance]; for mouse HA, clone 16B12 [BioLegend]), V5 (for rabbit V5, clone A190 [Bethyl]; for mouse V5, Invitrogen), and Myc (for rabbit Myc, clone Poly9063 [BioLegend]; for mouse Myc, clone 9E10 [BioLegend]).

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Software, FACS, Colorimetric Assay

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock.

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: Figure 1. E3 ubiquitin ligase TRIM21 mediates the degradation of HuR in response to heat shock (A) Immunoblot of lysates of MCF7 cells either unexposed or exposed to heat shock at 43C in presence of 10% FBS and collected at the indicated time points, probed with HuR and b Actin antibodies. Quantification of relative HuR protein levels (normalized to b Actin) with HuR protein level in cells not exposed to heat shock taken as 1. Quantification of relative fold change in HuR mRNA levels of MCF7 cells exposed to heat shock for the indicated time points with HuR mRNA level in cells not exposed to heat shock taken as 1. GAPDH mRNA level was used to normalize HuR mRNA levels. The data represent mean G SD values from three independent experiments. (B) Fluorescent confocal microscopic time-lapse images (60X) of live cells expressing HuR WT- EGFP fusion protein imaged for a period of 2 h at 37C or 43C. The scale bar is 20 mm. (C) Immunoblot of lysates of cells treated with CHX at 37C or at 43C or with both CHX and MG132 at 43C and collected at the indicated time points, probed with antibodies against HuR and b Actin. (D) Quantification of relative HuR protein levels (normalized to b Actin) plotted against indicated time points for each experimental setup in (C). Data represent mean G SD values from three independent experiments.

Article Snippet: Antibodies HuR/ELAV1 mouse monoclonal antibody (3A2) Santa Cruz Biotechnology, Dallas, Texas, United States Cat #. sc-5261 TRIM21 rabbit monoclonal antibody (D101D) Cell Signaling Technology, Danvers, MA, USA Cat #.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock.

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: Figure 3. S100 and E101 residues of HuR are determinants of recognition by TRIM21 (A) Schematic representation of HuR WT and deletion constructs, indicating the 3 RNA recognition motifs (RRMs), nuclear localization signal (NLS), linker and hinge regions of HuR. (B) Immunoblot of lysates of cells expressing His-tagged HuR WT and various His-tagged deletion mutants either exposed or unexposed to heat shock at 43C for 2 h, probed with antibodies against His tag and b Actin. (C) Cells co-expressing TRIM21 with either His-tagged HuR WT or various deletion mutants were exposed to heat shock at 43C for 1 h in presence of MG132. Lysates were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His and b Actin antibodies. Left panels represent the input lysates probed with the same antibodies with HuR antibody. (D) Contact map of HuR residues with TRIM21 counterparts generated by MD simulation, contact being defined as a distance less than 10 A˚ between respective a-carbons of amino acids. (E) Structure of HuR RRM1-2 (PDB: 4EGL) showing the molecular distance between S100 and K182 and between R115 and K182 residues. (F) Still from the MD simulation showing the bound state of RRM1-2 domain of HuR (blue) with the PRYSPRY domain of TRIM21 (red). The zoomed-in image shows the close contact between S100 of HuR (licorice structure) and the PRYSPRY domain of TRIM21. See also Figures S9 and S10.

Article Snippet: Antibodies HuR/ELAV1 mouse monoclonal antibody (3A2) Santa Cruz Biotechnology, Dallas, Texas, United States Cat #. sc-5261 TRIM21 rabbit monoclonal antibody (D101D) Cell Signaling Technology, Danvers, MA, USA Cat #.

Techniques: Construct, Western Blot, Expressing, Generated

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock.

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: Figure 4. S100 and E101 residues of HuR determine TRIM21 binding and degradation of HuR under heat shock (A) Schematic representation of HuR WT and point mutants indicating the point mutations S100A, E101A, S100A/E101A double mutant and R115A/T116A double mutant. (B)Immunoblot oflysatesofMCF7 cellsexpressingeitherHis-tagged HuRWT orHuRE101A orHuRS100A orHuRS100A/E101A mutant exposed or unexposed to heat shock at 43C for 2 h, probed with antibodies against His tag and b Actin. (C) Lysates of cells expressing TRIM21 together with either His-tagged HuR WT or HuR E101A or HuR S100A or HuR S101A/E101A mutant proteins, exposed to heat shock at 43C for 1 h in presence of MG132, were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His and b Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (D) Lysates of cells expressing HA-tagged Ubiquitin together with either His-tagged HuR WT or HuR S100A/E101A mutant proteins, exposed to heat shock at 43C for 1 h in presence of MG132, were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with Ubiquitin, His, and b Actin antibodies. Left panels represent the input lysates

Article Snippet: Antibodies HuR/ELAV1 mouse monoclonal antibody (3A2) Santa Cruz Biotechnology, Dallas, Texas, United States Cat #. sc-5261 TRIM21 rabbit monoclonal antibody (D101D) Cell Signaling Technology, Danvers, MA, USA Cat #.

Techniques: Binding Assay, Mutagenesis, Western Blot, Expressing, Ubiquitin Proteomics

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock.

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: Figure 5. S100 phosphorylation is required for TRIM21 binding and degradation of HuR under heat shock (A) Lysates of MCF7 cells expressing His-tagged HuR WT or HuR S100A mutant proteins, exposed to heat shock at 43C for 1 h in presence of MG132, were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with phosphoserine, His and b Actin antibodies. Left panels represent the input lysates probed with His and b Actin antibodies. (B) Immunoblot of lysates of cells expressing His-tagged HuR WT or HuR S100D mutant proteins exposed or not exposed to heat shock at 43C for 2 h and probed with antibodies against His tag and b Actin. (C) Immunoblot of lysates of cells transfected with control siRNA or siTRIM21 together with His-tagged HuR S100D expression construct and not exposed to heat shock, probed with antibodies against TRIM21, His tag and b Actin. (D) Lysates of cells expressing TRIM21 and His-tagged HuR WT or HuR S100D mutant proteins, exposed to heat shock at 43C for 1 h in presence of MG132, were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His and b Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (E) Cells transfected with either empty vector (Mock), HuR WT or HuR S100D expression constructs were not exposed or exposed to heat shock at 43C for 2 h. MTT assay was performed immediately after 2 h of heat shock. Data represent mean G SD values from three independent experiments. (F) Cells transfected with either empty vector (Mock), HuR WT or HuR S100D expression constructs were unexposed or exposed to heat shock at 43C for 2 h. Caspase 3/7 activity assay was performed. Relative light unit values of each sample, representing caspase 3/7 activity, are expressed as fold change from that of mock transfected cells at 37C, taken as 1. Data represent mean G SD values from three independent experiments. *, #, $ signifies p value %0.05, **, ##, $$ signifies p value %0.01 and ***, ###, $$$ signifies p value %0.005 (paired two-tailed t-test) in all panels. See also Figure S16.

Article Snippet: Antibodies HuR/ELAV1 mouse monoclonal antibody (3A2) Santa Cruz Biotechnology, Dallas, Texas, United States Cat #. sc-5261 TRIM21 rabbit monoclonal antibody (D101D) Cell Signaling Technology, Danvers, MA, USA Cat #.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Mutagenesis, Western Blot, Transfection, Control, Construct, Plasmid Preparation, MTT Assay, Activity Assay, Two Tailed Test

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock.

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: Figure 6. AKT1 phosphorylates S100 leading to TRIM21-mediated degradation of HuR under heat shock (A) Phylogenetic analysis of HuR sequences from vertebrate species showing evolutionary conservation of AKT1 target consensus motif (RXXS/T) at S100. (B) Immunoblot of lysates of MCF7 cells treated with DMSO or 10 mM Akt1 inhibitor for 2 h and subsequently unexposed or exposed to heat shock at 43C for 2 h, probed with antibodies against HuR and b Actin. (C) Immunoblot of lysates of cells transfected with control siRNA or Akt1 siRNA, and unexposed or exposed to heat shock at 43C for 2 h, after 48 h of siRNA transfection, probed with antibodies against AKT1, HuR and b Actin. (D) Immunoblot of lysates of cells exposed to heat shock at 43C for the indicated time durations, probed with antibodies against phosphoAKT1, AKT1, HuR and b Actin. (E) Lysates of cells treated with MG132 for 1 h and unexposed or exposed to heat shock at 43C for 2 h, were immunoprecipitated with non-immune IgG and HuR antibody and the immunoprecipitates were probed with AKT1, phosphoAKT1, HuR and b Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (F) Cells were treated with DMSO or 10 mM Akt inhibitor for 2 h followed by 1 h MG132 treatment, and unexposed or exposed to heat shock at 43C for 2 h. Cell lysates were immunoprecipitated with non-immune IgG and HuR antibody and

Article Snippet: Antibodies HuR/ELAV1 mouse monoclonal antibody (3A2) Santa Cruz Biotechnology, Dallas, Texas, United States Cat #. sc-5261 TRIM21 rabbit monoclonal antibody (D101D) Cell Signaling Technology, Danvers, MA, USA Cat #.

Techniques: Western Blot, Transfection, Control, Immunoprecipitation

Journal: iScience

Article Title: An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock.

doi: 10.1016/j.isci.2023.106307

Figure Lengend Snippet: Figure 7. Concerted action by AKT1 and TRIM21 causes HuR degradation under heat shock (A) Cells were co-transfected with TRIM21 and His-tagged HuR WT expression constructs, treated with 10 mM Akt Inhibitor for 2 h followed by 1 h MG132 treatment, and unexposed or exposed to heat shock at 43C for 2 h. Cell lysates were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His and b Actin antibodies. Left panels represent the input lysates probed with the same antibodies. (B) Cells were co-transfected with TRIM21 and His-tagged HuR WT expression constructs along with control siRNA or Akt1 siRNA. After 46 h of transfection and 1 h of MG132 treatment, cells were unexposed or exposed to heat shock at 43C for 1 h. Lysates were subjected to Ni-NTA affinity pulldown and the precipitates were immunoblotted with TRIM21, His, b Actin and AKT1 antibodies. Left panels represent the input lysates probed with the same antibodies. (C) Lysates of cells expressing TRIM21 together with His-tagged HuR WT or HuR R97A proteins and treated with MG132 and unexposed or exposed to heat shock at 43C for 1 h, were subjected to Ni-NTA affinity pulldown. The precipitates were probed with antibodies against TRIM21, His and b Actin. Left panels represent the input lysates probed with the same antibodies. (D) In vitro ubiquitination of p-Ser HuR by TRIM21. Purified His-tagged HuR was either untreated or treated with purified AKT1 protein to cause serine phosphorylation. The serine-phosphorylated HuR was further purified using Ni-affinity

Article Snippet: Antibodies HuR/ELAV1 mouse monoclonal antibody (3A2) Santa Cruz Biotechnology, Dallas, Texas, United States Cat #. sc-5261 TRIM21 rabbit monoclonal antibody (D101D) Cell Signaling Technology, Danvers, MA, USA Cat #.

Techniques: Transfection, Expressing, Construct, Control, In Vitro, Ubiquitin Proteomics, Phospho-proteomics

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: Colocalization of host DNA replication factors with B19V replication centers using immunofluorescence assay and proximity ligation assay (PLA). CD36+ EPCs were infected with B19V. At 24 hpi, cells were cytospun onto slides. (A) Immunofluorescence assay was performed to stain replicating viral genome (BrdU labeled), RPA32, and one of the host DNA replication proteins, Pol δ, Pol α, Pol ε, PCNA, or RFC1, as indicated. STED superresolution microscopy images were taken using a Leica TCS SP8 STED microscope under a 100× objective lens. (B) PLA was performed by costaining the cells with mouse anti-BrdU antibody and one of the rabbit antibodies against Pol δ, Pol α, Pol ε, PCNA, and RFC1, as indicated, which produces amplified signals for interacted proteins in close proximity. Confocal images were taken using an Eclipse C1 Plus confocal microscope (Nikon) that was controlled by Nikon EZ-C1 software. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Immunofluorescence, Proximity Ligation Assay, Infection, Staining, Labeling, Microscopy, Amplification, Software

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: Pol δ and RPA32 play an important role in B19V DNA replication. (A) Western blot analysis of shRNA knockdown. CD36+ EPCs transduced with shRNA-expressing lentiviruses, as indicated, were collected at 2 days postransduction. The cells were analyzed for expression of Pol α, Pol δ, Pol ε, Pol η, Pol λ, and RPA32, as indicated, by Western blotting. β-Actin was used as a loading control. (B) Cell cycle analysis. At 2 days postransduction, shRNA-transduced CD36+ EPCs were labeled with BrdU and analyzed for cell proliferation using flow cytometry with anti-BrdU and DAPI costaining. The percentages of the cells in each phase of the cell cycle are shown as means ± standard deviations (STD) of data obtained from three independent experiments. (C) Southern blot analysis of B19V DNA replication. CD36+ EPCs were transduced with the indicated shRNA for 2 days, and the cells were infected with B19V. At 48 hpi, Hirt DNA was extracted from infected cells and analyzed by Southern blotting with the M20 DNA probe (upper) and the mitochondrial DNA (Mito DNA) probe (lower). (D) B19V progeny virion production. CD36+ EPCs were transduced with the indicated shRNAs for 2 days, and the cells were infected with B19V. After 3 h of incubation, the infected cells were washed twice with phosphate-buffered saline (PBS) and cultured with fresh medium. At 48 hpi, both cells and medium were collected for quantification of B19V virions using qPCR. The value for virus (vgc) produced from shScram-transduced CD36+ EPCs is arbitrarily set at 100. **, P < 0.005; ***, P < 0.001; n.s., not significant.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Western Blot, shRNA, Transduction, Expressing, Cell Cycle Assay, Labeling, Flow Cytometry, Southern Blot, Infection, Incubation, Cell Culture, Produced

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: All forms of phosphorylated RPA32 are expressed in B19V-infected cells. CD36+ EPCs were infected with B19V or mock infected. At 48 hpi, the cells were collected and lysed for Western blotting of total RPA32 (A) and of various forms of phosphorylated RPA32 as indicated (B). β-Actin was used as a loading control.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Infection, Western Blot

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: All forms of phosphorylated RPA32 are associated with the BrdU-labeled replicating viral genome. CD36+ EPCs were infected with B19V. At 24 hpi, the cells were labeled with BrdU and collected for immunofluorescence (IF) assay with antibodies against RPA32(S4/8), RPA32(T21), RPA32(S33), total RPA32, and anti-BrdU. (A) Images were taken using a Leica TCS SPE confocal microscope at ×63 magnification. (B and C) (B) Images were taken using a Leica TCS SP8 STED superresolution microscope under a 100× objective lens and processed with deconvolution editing using Huygens software. (C) Quantification of the intensity of each stained signaling was performed using Huygens Software.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Labeling, Infection, Immunofluorescence, Microscopy, Software, Staining

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: Both phosphorylated and unphosphorylated RPA32 proteins play a role in B19V replication. (A) Expression of RPA32 mutants. Flag-tagged wild-type RPA32 and phosphorylation mutants, namely, RPA32(S4/8A, S4/8D, S33A, S33D, S4/8T21S33 to A or D mutations [all A or D]), were expressed in UT7/Epo-S1 cells by lentivirus transduction. Endogenous RPA32 was knocked down in these cell lines by transducing a shRPA32-expressing lentivirus. Protein expression of RPA32 mutants was detected using anti-RPA32 or anti-Flag antibodies. β-Actin was used as a loading control. Arrowheads indicate endogenous unphosphorylated RPA32. (B) Southern blot analysis of B19V DNA replication. RPA32 mutant-expressing UT7/Epo-S1 cells were cultured under hypoxia conditions and electroporated with SalI-linearized pM20. At 48 hpi, Hirt DNA was extracted from the transfected cells, digested with DpnI enzyme, and analyzed by Southern blotting using the M20 probe (upper panel) and the mitochondrial (Mito) DNA probe (lower panel). (C) B19V virion production. RPA32 mutant-expressing UT7/Epo-S1 cells were cultured under hypoxia conditions for 2 days and electroporated with SalI-linearized M20. At 48 hpi, the cell samples were collected and quantified for B19V virions using qPCR. The value for virus (vgc) produced from M20-transfected RPA32(WT)-expressing UT7/Epo-S1 cells was arbitrarily set at 100. n.s., not significant.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Expressing, Transduction, Southern Blot, Mutagenesis, Cell Culture, Transfection, Produced

Journal: medRxiv

Article Title: Identifying risk genes for embryo aneuploidy using ultra-low coverage whole-genome sequencing

doi: 10.1101/2023.07.22.23292618

Figure Lengend Snippet: (A) Expression and localization of CCDC66 in different stages of mouse oocyte meiotic maturation. Oocytes were stained with anti-CCDC66 antibody (gray) at (0, 5, 7, and 16) hours of maturation corresponding to Prophase I, Pro-metaphase I, Metaphase I and Metaphase II, respectively. Tubulin and DAPI was used to label the spindle and DNA (green and blue, respectively). (B) Prophase-I arrested oocytes were co-microinjected with Trim21 cRNA and either CCDC66 antibody or IgG. Oocytes were fixed at metaphase I and stained to detect CCDC66 (gray). Tubulin (green) and DAPI (blue) were used to label the spindle and DNA. (C) Relative CCDC66 intensity from B, two-tailed unpaired Students t-Test (***p < 0.001). (D) Quantification of percentage of polar body extrusion (PBE). (E) Percentage of aneuploid Metaphase II eggs after knockdown of CCDC66 in young and old mouse oocytes (One-way ANOVA, * p <0.05; ** p < 0.01, *** p < 0.001). Number of oocytes examined: young IgG: 34, young CCDC66: 37, old IgG: 29, old CCDC66: 31. These experiments were repeated 3 times. Scale bars: 15 and 4 μm (insets).

Article Snippet: Rabbit anti-CCDC66 antibody (Bethyl Laboratories, #A303-339A) and control IgG antibody (Merck Millipore, #12-370) were purified using Amicon Ultra 0.5-ml Centrifugal Filter (Merk Millipore, #UFC5003096). pGEMHE-Cherry-TRIM21 (Addgene, #105522) or pGEMHE-mEGFP-mTrim21 (Addgene, #105519) were linearized with Asc I (New England Biolabs, #R0558S,) and in vitro transcribed using a T7 mMessage mMachine Kit (Ambion, #AM1340).

Techniques: Expressing, Staining, Two Tailed Test