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: Oncogene

Article Title: TAF1 acetyltransferase promotes colorectal carcinoma metastasis by catalyzing β-hydroxybutyrylation of KCTD9

doi: 10.1038/s41388-025-03644-1

Figure Lengend Snippet: A Tubacin decreases KCTD9 protein and increases KCTD9 K123bhb and K129bhb levels in HEK-293T and different CRC cell lines. HEK293T, HCT116, and LOVO cells were treated with Tubacin for the indicated times. Cell lysates were immunoprecipitated with anti-KCTD9 and analyzed by WB. B Tubacin has a negligible effect on KCTD9 mRNA levels. HEK293T, HCT116, and LOVO cells were treated with Tubacin for the indicated times. The relative KCTD9 mRNA levels were determined by qPCR. C , D Immunoblot and qPCR analysis of the KCTD9 K123bhb and K129bhb ( C ), and KCTD9 mRNA levels ( D ) in control and TAF1-knockdown HCT116 and LOVO cells. E Co-IP of endogenous TRIM21 with anti KCTD9 antibody in HCT116 cells. F Effects of TAF1 WT or HAT activity-deficient mutant Δ844-850 on KCTD9 association with TRIM21. Myc-TRIM21 and indicated Flag-TAF1 were co-transfected into HEK-293T cells with HA-KCTD9 constructs or empty vector control (EV). G Flag-tagged TRIM21 protein ubiquitinated HA-tagged KCTD9 analyzed by in vitro ubiquitination assays using purified recombinant proteins. Purified recombinant TRIM21 and TRIM21 ΔRING protein were incubated with ATP, E1, E2 proteins, ubiquitin, KCTD9-WT peptide and KCTD9-Kbhb peptide (10 μM or 20 μM) along with purified recombinant HA-tagged KCTD9 protein. H , I Effects of ectopic expression of KCTD9 WT or KCTD9 2KR mutant on KCTD9 protein degradation in HCT116 cells co-transfection of Myc-TRIM21 and Flag-TAF1 in exposure to cycloheximide (CHX, 100 μM) treatment (0, 30, 60, and 90 min). J Effects of KCTD9 WT, 2KQ or 2KR mutant on KCTD9 ubiquitination in HCT116/sgKCTD9 cells co-transfection of Myc-TRIM21 or/and Flag-TAF1. Data are representative of two or three independent experiments with similar results. Error bars, ± SD. * P < 0.05 or ** P < 0.01, by paired two-way Student’s t-test. n.s., negative significant.

Article Snippet: Antibodies for TAF1 (#12781), HES1 (#11988), CXCR4 (#64837), CXCL12 (#3740), activated Notch1 (NICD, #4380), CBP (#7389 T), P300 (#86377), TRIM21 (#92043), HA (#3724), Actin (#4967S) and His (#12698) were from CST (Danvers, MA); anti-β-Hydroxybutyryllysine (#PTM-1201RM) was from PTM BIO (Chicago, USA); antibody for KCTD9 (ab180937) were from Abcam Biotechnology (Abcam, Cambridge, UK); HEY1 (#19929-1-AP), MYC (#10828-1-AP), SNAI1 (#13099-1-AP) were from Proteintech; and anti-KCTD9 K123bhb and K129bhb rabbit polyclonal antibody generated by Proteintech using the peptide DKGVWGN-K(bhb)-QDHRGAF and D-K(bhb)-GVWGNK; and KCTD9-Kbhb peptide was synthesized by using the peptide of DK(bhb)GVWGN-K(bhb)-QDHRGAF; an antibody for FLAG (F3165) was from Sigma-Aldrich; antibodies for GAPDH (sc-25778) and GST (sc-138) were from Santa Cruz Biotechnology (Santa Cruz, CA); the secondary antibodies, anti-rabbit IgG, HRP-linked antibody (#7074) and anti-mouse IgG, HRP-linked antibody (#7076) were purchased from CST (Danvers, MA).

Techniques: Immunoprecipitation, Western Blot, Control, Knockdown, Co-Immunoprecipitation Assay, Activity Assay, Mutagenesis, Transfection, Construct, Plasmid Preparation, In Vitro, Ubiquitin Proteomics, Purification, Recombinant, Incubation, Expressing, Cotransfection

Journal: Oncogene

Article Title: TAF1 acetyltransferase promotes colorectal carcinoma metastasis by catalyzing β-hydroxybutyrylation of KCTD9

doi: 10.1038/s41388-025-03644-1

Figure Lengend Snippet: A Representative images of TAF1, KCTD9 K123bhb, KCTD9 K129bhb and NICD expressions in 113 clinical CRC primary tumor tissues. Scale bars, 50 μm. B Correlation of expression levels between TAF1, KCTD9 K123bhb, KCTD9 K129bhb and NICD in ( A ). C Prognosis comparison of CRC patients with TAF1/KCTD9-K123bhb, TAF1/KCTD9-K129bhb and TAF1/NICD ectopic differential expression using Kaplan-Meier survival analysis. D A working model of TAF1 promoted CRC metastasis through the KCTD9 Kbhb modification status. TAF1 directly binds to and catalyzes KCTD9 Kbhb modification at lys123 and lys129, thereby enhancing KCTD9 association with the E3 ubiquitin ligase TRIM21. This interaction leads to the promotion of KCTD9 degradation via the ubiquitin-proteasome pathway. KCTD9, as a tumor suppressor, its low levels activated Notch signaling pathway, ultimately contributes to the CRCSC properties, the progression of CRC and liver metastasis.

Article Snippet: Antibodies for TAF1 (#12781), HES1 (#11988), CXCR4 (#64837), CXCL12 (#3740), activated Notch1 (NICD, #4380), CBP (#7389 T), P300 (#86377), TRIM21 (#92043), HA (#3724), Actin (#4967S) and His (#12698) were from CST (Danvers, MA); anti-β-Hydroxybutyryllysine (#PTM-1201RM) was from PTM BIO (Chicago, USA); antibody for KCTD9 (ab180937) were from Abcam Biotechnology (Abcam, Cambridge, UK); HEY1 (#19929-1-AP), MYC (#10828-1-AP), SNAI1 (#13099-1-AP) were from Proteintech; and anti-KCTD9 K123bhb and K129bhb rabbit polyclonal antibody generated by Proteintech using the peptide DKGVWGN-K(bhb)-QDHRGAF and D-K(bhb)-GVWGNK; and KCTD9-Kbhb peptide was synthesized by using the peptide of DK(bhb)GVWGN-K(bhb)-QDHRGAF; an antibody for FLAG (F3165) was from Sigma-Aldrich; antibodies for GAPDH (sc-25778) and GST (sc-138) were from Santa Cruz Biotechnology (Santa Cruz, CA); the secondary antibodies, anti-rabbit IgG, HRP-linked antibody (#7074) and anti-mouse IgG, HRP-linked antibody (#7076) were purchased from CST (Danvers, MA).

Techniques: Expressing, Comparison, Quantitative Proteomics, Modification, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Opposing roles of E3 ligases TRIM23 and TRIM21 in regulation of ion channel ANO1 protein levels

doi: 10.1016/j.jbc.2021.100738

Figure Lengend Snippet: TRIM21 destabilizes ANO1. A , left , endogenous ANO1 was more markedly decreased after hemagglutinin–TRIM21 expression in TRIM23 KO than WT ZR-75-1 cells. Long and short exposures ( top half ) clearly show TRIM21 effect in TRIM23 WT and KO cells, respectively. GAPDH, loading control. Right , summary data of the left panel. ∗∗ p = 0.0011, n = 5. ANO1 expression was decreased in TRIM23 KO cells. Data are normalized to ANO1 expression without hemagglutinin–TRIM21 expression. B , left , TRIM21 knockdown through RNAi robustly elevated ANO1 expression in ZR-75-1 cells. Right , summary data of the left panel . ∗ p = 0.03 for ANO1, ∗∗ p = 0.005 for TRIM21, n = 4. C , TRIM21 KO increased ANO1 protein but not mRNA level in both the salivary gland and heart relative to the level in sex-matched WT littermates. GFP: reporter of Trim21 transcription in TRIM21 KO mice. Middle , summary data of the left panel ; right , summary data of mRNA levels. ∗ p ≤ 0.033 (n = 7 for the salivary gland, n = 5 for the heart). For mRNA level, p = 0.640, n = 3 for the salivary gland; p = 0.041, n = 3 for the heart. D , TRIM21 KO increased saliva secretion in mice. ∗ p = 0.032, n = 4 (WT/HE: two WT and two heterozygous). E , the heart rate did not differ significantly between TRIM21 KO mice and their WT/HE littermates; p = 0.259, n = 5 (WT/HE: three WT and two heterozygous). ANO1, anoctamin-1; Ctrlsh, control shRNA; RNAi, RNA interference; TRIM, tri partite m otif; TRIM21sh, TRIM21 shRNA.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-human ANO1 (ab64085) and rabbit polyclonal anti-mouse ANO1 (ab53212), Abcam; rabbit anti-human/mouse TRIM23 (HPA-039605) and mouse anti-FLAG (F1804), Sigma-Aldrich; mouse anti-HA (MMS-101P), Covance Research Products Inc; mouse anti-V5 (P/N-46-0705) and anti-Xpress (R910-25), Invitrogen; and mouse anti-human TRIM21 (sc-25351), anti-ubiquitin (sc-8017), anti-GAPDH (sc-365062), and anti-actin (sc-47778), Santa Cruz Biotechnology.

Techniques: Expressing, Control, Knockdown, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Opposing roles of E3 ligases TRIM23 and TRIM21 in regulation of ion channel ANO1 protein levels

doi: 10.1016/j.jbc.2021.100738

Figure Lengend Snippet: ANO1 physically interacts with TRIM21. A , human ANO1–V5 physically and functionally interacts with human Xpress–TRIM21 in HEK293T cells. Anti-Xpress antibody pulled down ANO1 together with Xpress–TRIM21 from cells coexpressing ANO1–V5 and Xpress-TRIM21 but not from cells expressing ANO1–V5 alone. TRIM21 expression also moderately lowered ANO1 protein expression and increased ANO1 ubiquitination to a limited extent. In IP, the asterisk denotes IgG; the bracket denotes TRIM21 doublet also detected in “Input.” B , exogenous Xpress–TRIM21 in HEK293T cells was captured by GST–ANO1C but not GST alone. Results shown are representative of three independent biological replicates. The asterisk denotes undegraded GST or GST–ANO1. C , endogenous TRIM21 ( red ∗) and ANO1 ( black ∗) interact in ZR-75-1 cells. Upper , endogenous ANO1 was immunoprecipitated with anti-mouse ANO1 or control IgG and then immunoblotted with anti-ANO1 ( top ) and anti-TRIM21 ( bottom ). Lower , endogenous TRIM21 was immunoprecipitated with anti-TRIM21 or control IgG and then immunoblotted with anti-ANO1 ( top ) and anti-TRIM21 ( bottom ). All data in panels A – C are representative of three independent biological replicates. ANO1, anoctamin-1; ANO1–V5, V5-tagged full-length ANO1; ANO1C, ANO1 C terminus; Ig G, immunoglobulin G; TRIM, tri partite m otif; Xpress-TRIM21, Xpress-tagged TRIM21.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-human ANO1 (ab64085) and rabbit polyclonal anti-mouse ANO1 (ab53212), Abcam; rabbit anti-human/mouse TRIM23 (HPA-039605) and mouse anti-FLAG (F1804), Sigma-Aldrich; mouse anti-HA (MMS-101P), Covance Research Products Inc; mouse anti-V5 (P/N-46-0705) and anti-Xpress (R910-25), Invitrogen; and mouse anti-human TRIM21 (sc-25351), anti-ubiquitin (sc-8017), anti-GAPDH (sc-365062), and anti-actin (sc-47778), Santa Cruz Biotechnology.

Techniques: Expressing, Ubiquitin Proteomics, Immunoprecipitation, Control

Journal: The Journal of Biological Chemistry

Article Title: Opposing roles of E3 ligases TRIM23 and TRIM21 in regulation of ion channel ANO1 protein levels

doi: 10.1016/j.jbc.2021.100738

Figure Lengend Snippet: Schematic showing ANO1 regulation by antagonistic TRIM23 and TRIM21 E3 ligases. Both TRIM23 and TRIM21 bind to ANO1 C terminus. TRIM23 ubiquitinates and stabilizes ANO1 by preventing lysosomal degradation, whereas TRIM21 ubiquitinates and causes ANO1 degradation. EGF signaling enhances ANO1 expression through TRIM23 by a mechanism that remains unclear but potentially involves blocking of TRIM21 ubiquitination of ANO1 (rather than blocking of TRIM21 to the same site—ANO1 C terminus). Green and red dots , antiproteolytic and proteolytic ubiquitination. ANO1, anoctamin-1; EGF, epidermal growth factor; N and C, N- and C-termini; TRIM, tri partite m otif.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-human ANO1 (ab64085) and rabbit polyclonal anti-mouse ANO1 (ab53212), Abcam; rabbit anti-human/mouse TRIM23 (HPA-039605) and mouse anti-FLAG (F1804), Sigma-Aldrich; mouse anti-HA (MMS-101P), Covance Research Products Inc; mouse anti-V5 (P/N-46-0705) and anti-Xpress (R910-25), Invitrogen; and mouse anti-human TRIM21 (sc-25351), anti-ubiquitin (sc-8017), anti-GAPDH (sc-365062), and anti-actin (sc-47778), Santa Cruz Biotechnology.

Techniques: Expressing, Blocking Assay, Ubiquitin Proteomics

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: 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: 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