Journal: bioRxiv

Article Title: Hypoxia-mediated regulation of DDX5 through decreased chromatin accessibility and post-translational targeting restricts R-loop accumulation

doi: 10.1101/2022.04.30.490097

Figure Lengend Snippet: A. RKO cells were exposed to hypoxia (Hyp, <0.1% O 2 ) for the times shown and mRNA expression of TRIM5 , TRIM21 and VEGFA was analyzed by qPCR and normalized to 18S housekeeping gene. Statistical significance was determined with two-tailed student t-test for each gene by comparing control and specific time in hypoxia (* P <0.05, *** P <0.001, *** P <0.0001, ns non-significant). B-C . RKO and HCT116 cells were exposed to hypoxia for the times indicated and subjected to western blotting with TRIM5 and DDX5 antibodies. D. Expression of DDX5 and TRIM5 proteins was analyzed in non-malignant (labeled in green) and cancer cell lines (labeled in red) as indicated, by western blotting. Histone H3 and β-actin were used as loading controls. E. HCT116 cells were exposed to <0.1 or 2% O 2 for the times indicated and were subjected to western blotting with the antibodies indicated. F-G . OE21 cells were exposed to hypoxia for the times indicated and subjected to a qPCR (G) and western blotting (H) analysis. 18S served as a housekeeping gene in the qPCR analysis. H. HCT116 p53+/+ and p53−/− cells were exposed to 18 h of hypoxia (Hyp, <0.1% O 2 ) and TRIM5 expression was analysed relative to 18S. INPP5D expression is shown as a positive control for p53-dependent hypoxia-inducible target gene. Bar graphs show mean expression and SEM of 3 biological replicates. Statistical significance was determined with two-tailed student t-test for each gene and conditions indicated (* P <0.05, ** P <0.01). I . HCT116 cells were exposed to 6 h of hypoxia (<0.1% O 2 ) or non-treated (Norm) and lysates were subjected to immunoprecipitation (IP) with polyclonal goat anti-TRIM5 antibody or goat IgG control. TRIM5 and DDX5 interaction was determined in IP samples by western blotting and compared to the input, as shown. J . HCT116 cells were transfected with non-targeting siRNA control (Scr), two different siRNA duplexes against TRIM5 (siTRIM5#1 and siTRIM5#2) or were Mock transfected (no siRNA) and the next day cells were exposed to 16 h of hypoxia (<0.1% O 2 ). Protein expression was analyzed by western blotting.

Article Snippet: Cellular extracts were incubated with TRIM5 antibody (AF3684, R&D systems) or IgG control (Millipore) with end-to-end rotation overnight at 4°C.

Techniques: Expressing, Two Tailed Test, Control, Western Blot, Labeling, Positive Control, Immunoprecipitation, Transfection

Journal: Cells

Article Title: Association Analysis of Single-Cell RNA Sequencing and Proteomics Reveals a Vital Role of Ca 2+ Signaling in the Determination of Skeletal Muscle Development Potential

doi: 10.3390/cells9041045

Figure Lengend Snippet: Top-10 upregulated and downregulated proteins.

Article Snippet: The membranes were then incubated with primary antibodies overnight at 4 °C against GAPDH (CSB-PA00025A0Rb), ABCB1 (CSB-PA11737A0Rb), AKR1B1 (CSB-PA001539LA01HU), ALDH1A1 (CSB-PA001565LA01MO), PRMT3 (CSB-PA018733LA01HU), TRIM5 (CSB-PA874861LA01HU), TSTA3 (CSB-PA00935A0Rb), and YARS2 (CSB-PA903762) (1:500 dilution, CUSABIO, Wuhan, China).

Techniques: Binding Assay, Transplantation Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

doi: 10.1074/jbc.m111.260406

Figure Lengend Snippet: FIGURE 1. The RING domain is required for TRIM5 higher order self-association. A, the arrangement of the domains of TRIM5 and the other TRIM proteinsusedinthisstudyisshown.B,celllysatesfrom293TcellstransientlytransfectedwithplasmidsexpressingHA-taggedwild-typeTRIM5rhortheRING and RING-L1 mutants were cross-linked with increasing amounts of glutaraldehyde (GA; final concentrations 0, 0.4, 1, 2, and 4 mM). The cross-linked products were resolved by SDS-PAGE and visualized by Western blotting with an anti-HA antibody. The positions of the molecular weight markers are indicated to the left of the blot. C, coprecipitation of HA-tagged TRIM5rh variants with FLAG-tagged TRIM5rh dimers. 293T cells were transiently transfected with the empty pLPCX vector or vectors expressing the indicated TRIM5rh variants. Cytosolic extracts containing the wild-type and mutant TRIM5rh dimers were prepared separately, mixed in a 1:1 ratio, and used for precipitation with an anti-FLAG antibody. The amount of HA- and FLAG-tagged proteins in the lysates (Input) and immunoprecipitates (Pellet) was analyzed by Western blotting with HRP-conjugated anti-HA and anti-FLAG antibodies. D, HIV-1 CA-NC binding assay. Cell lysates from 293T cells transiently expressing wild-type TRIM5rh and TRIM5rh RING and RING-L1 mutants were used in the HIV-1 CA-NC binding assay. The TRIM5rh RING and RING-L1 mutants are designated R and R-L1, respectively. Two different input concentrations of the RING-L1 mutant were used in the assay. The top panel shows the input TRIM5rh proteins; the middle panel shows TRIM5 proteins that co-sediment through the 70% sucrose cushion with the assembled HIV-1 CA-NC complexes. The HIV-1 CA-NC complexes in the pellets were detected by Western blotting with an antibody directed against the p24 CA protein (bottom panel). E, expression levels of the wild-type TRIM5rh and RING and RING-L1 mutants in stable chicken DF1 cell lines. Western blotting was done with a polyclonal anti-TRIM5 antibody (Imgenex) and an anti--actin antibody as control. F and G, DF1 cells stably expressing the empty vector pLPCX, wild-type TRIM5rh, RING, and RING-L1 were incubated with various amounts of HIV-1-GFP (F) and N-MLV-GFP viruses (G). Infected GFP- positive cells were counted by FACS.

Article Snippet: Expression of the TRIM5 proteins was confirmed by Western blotting with an anti-TRIM5 polyclonal antibody (1:2000; Imgenex), followed by an HRP-conjugated anti-rabbit secondary antibody (1:1000; Sigma).

Techniques: SDS Page, Western Blot, Molecular Weight, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Binding Assay, Control, Stable Transfection, Incubation, Infection

Journal: Journal of Biological Chemistry

Article Title: Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

doi: 10.1074/jbc.m111.260406

Figure Lengend Snippet: FIGURE5.ContributionofTRIM5dimerizationtohigherorderself-asso- ciation. A, coprecipitation of HA-tagged TRIM5rh coiled-coil domain mutants with FLAG-tagged wild-type TRIM5rh dimers. 293T cells were tran- siently transfected with plasmids encoding HA-tagged wild-type TRIM5rh or TRIM5rh with alterations in the coiled-coil domain. Cytosolic extracts from these cells were mixed in a 1:1 ratio with lysates from 293T cells transiently transfected with the empty vector pLPCX () or the plasmid expressing FLAG-tagged wild-type TRIM5rh (). The mixture was precipitated with an anti-FLAG antibody. The abilities of these coiled-coil domain mutants to dimerize and their co-IP indices are indicated below the Western blots. B, coprecipitation of HA-tagged wild-type TRIM5rh by FLAG-tagged C-termi- nal deletion mutants of TRIM5. RBCC-L2 is TRIM5rh with a deletion of the B30.2(SPRY) domain; RBCC is TRIM5rh with a deletion of both the B30.2(SPRY) domain and the Linker 2 region.

Article Snippet: Expression of the TRIM5 proteins was confirmed by Western blotting with an anti-TRIM5 polyclonal antibody (1:2000; Imgenex), followed by an HRP-conjugated anti-rabbit secondary antibody (1:1000; Sigma).

Techniques: Transfection, Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

doi: 10.1074/jbc.m111.260406

Figure Lengend Snippet: FIGURE 7. Higher order association between wild-type TRIM5rh, TRIM4hu, and chimeras with individual replacement of the RING or the B-box 2 domain. A, schematic diagram of wild-type TRIM5rh, TRIM4hu, and the chimeras. B, coprecipitation of wild-type TRIM5rh, TRIM5rh with the TRIM4 RING or B-box 2 domain (4R and 4B, respectively), and TRIM4 with the TRIM5rh RING or B-box 2 domains (5R and 5B, respectively). C, coprecipita- tion of wild-type TRIM4hu or TRIM4hu with the TRIM5rh RING or B-box 2 domain (5R and 5B, respectively) and TRIM5 with the TRIM4 RING or B-box 2 domain (4R and 4B, respectively). The co-IP indices are indicated below the Western blots.

Article Snippet: Expression of the TRIM5 proteins was confirmed by Western blotting with an anti-TRIM5 polyclonal antibody (1:2000; Imgenex), followed by an HRP-conjugated anti-rabbit secondary antibody (1:1000; Sigma).

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

doi: 10.1074/jbc.m111.260406

Figure Lengend Snippet: FIGURE 8. Higher order associations of the TRIM5rh/TRIM4hu chimeras with replacements of both the RING and the B-box 2 domains. A, sche- maticdiagramofwild-typeTRIM5rhandTRIM4huandthechimeras.B,higher order association of the RING-B-box 2 (RB) chimeras, the B30.2(SPRY) domain chimera (4–5SPRY), and the wild-type parental TRIM5 and TRIM4 proteins. The 4RB chimera is a TRIM5rh protein in which both the RING and B-box 2 domains have been replaced by those of TRIM4. The 5RB chimera is a TRIM4 protein in which both the RING and B-box 2 domains have been replaced by those of TRIM5. The 4–5SPRY chimera is a TRIM4 protein with the B30.2(SPRY) domain replaced by that of TRIM5. The co-IP indices of the chimeras were normalizedtothoseassociatedwithTRIM4self-associationand/orTRIM5self- association and are indicated below the Western blots.

Article Snippet: Expression of the TRIM5 proteins was confirmed by Western blotting with an anti-TRIM5 polyclonal antibody (1:2000; Imgenex), followed by an HRP-conjugated anti-rabbit secondary antibody (1:1000; Sigma).

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

doi: 10.1074/jbc.m111.260406

Figure Lengend Snippet: FIGURE 1. The RING domain is required for TRIM5 higher order self-association. A, the arrangement of the domains of TRIM5 and the other TRIM proteinsusedinthisstudyisshown.B,celllysatesfrom293TcellstransientlytransfectedwithplasmidsexpressingHA-taggedwild-typeTRIM5rhortheRING and RING-L1 mutants were cross-linked with increasing amounts of glutaraldehyde (GA; final concentrations 0, 0.4, 1, 2, and 4 mM). The cross-linked products were resolved by SDS-PAGE and visualized by Western blotting with an anti-HA antibody. The positions of the molecular weight markers are indicated to the left of the blot. C, coprecipitation of HA-tagged TRIM5rh variants with FLAG-tagged TRIM5rh dimers. 293T cells were transiently transfected with the empty pLPCX vector or vectors expressing the indicated TRIM5rh variants. Cytosolic extracts containing the wild-type and mutant TRIM5rh dimers were prepared separately, mixed in a 1:1 ratio, and used for precipitation with an anti-FLAG antibody. The amount of HA- and FLAG-tagged proteins in the lysates (Input) and immunoprecipitates (Pellet) was analyzed by Western blotting with HRP-conjugated anti-HA and anti-FLAG antibodies. D, HIV-1 CA-NC binding assay. Cell lysates from 293T cells transiently expressing wild-type TRIM5rh and TRIM5rh RING and RING-L1 mutants were used in the HIV-1 CA-NC binding assay. The TRIM5rh RING and RING-L1 mutants are designated R and R-L1, respectively. Two different input concentrations of the RING-L1 mutant were used in the assay. The top panel shows the input TRIM5rh proteins; the middle panel shows TRIM5 proteins that co-sediment through the 70% sucrose cushion with the assembled HIV-1 CA-NC complexes. The HIV-1 CA-NC complexes in the pellets were detected by Western blotting with an antibody directed against the p24 CA protein (bottom panel). E, expression levels of the wild-type TRIM5rh and RING and RING-L1 mutants in stable chicken DF1 cell lines. Western blotting was done with a polyclonal anti-TRIM5 antibody (Imgenex) and an anti--actin antibody as control. F and G, DF1 cells stably expressing the empty vector pLPCX, wild-type TRIM5rh, RING, and RING-L1 were incubated with various amounts of HIV-1-GFP (F) and N-MLV-GFP viruses (G). Infected GFP- positive cells were counted by FACS.

Article Snippet: Western blotting was done with a polyclonal anti-TRIM5 antibody (Imgenex) and an anti- -actin antibody as control.

Techniques: SDS Page, Western Blot, Molecular Weight, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Binding Assay, Control, Stable Transfection, Incubation, Infection

Journal: Journal of Biological Chemistry

Article Title: Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

doi: 10.1074/jbc.m111.260406

Figure Lengend Snippet: FIGURE5.ContributionofTRIM5dimerizationtohigherorderself-asso- ciation. A, coprecipitation of HA-tagged TRIM5rh coiled-coil domain mutants with FLAG-tagged wild-type TRIM5rh dimers. 293T cells were tran- siently transfected with plasmids encoding HA-tagged wild-type TRIM5rh or TRIM5rh with alterations in the coiled-coil domain. Cytosolic extracts from these cells were mixed in a 1:1 ratio with lysates from 293T cells transiently transfected with the empty vector pLPCX () or the plasmid expressing FLAG-tagged wild-type TRIM5rh (). The mixture was precipitated with an anti-FLAG antibody. The abilities of these coiled-coil domain mutants to dimerize and their co-IP indices are indicated below the Western blots. B, coprecipitation of HA-tagged wild-type TRIM5rh by FLAG-tagged C-termi- nal deletion mutants of TRIM5. RBCC-L2 is TRIM5rh with a deletion of the B30.2(SPRY) domain; RBCC is TRIM5rh with a deletion of both the B30.2(SPRY) domain and the Linker 2 region.

Article Snippet: Western blotting was done with a polyclonal anti-TRIM5 antibody (Imgenex) and an anti- -actin antibody as control.

Techniques: Transfection, Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

doi: 10.1074/jbc.m111.260406

Figure Lengend Snippet: FIGURE 7. Higher order association between wild-type TRIM5rh, TRIM4hu, and chimeras with individual replacement of the RING or the B-box 2 domain. A, schematic diagram of wild-type TRIM5rh, TRIM4hu, and the chimeras. B, coprecipitation of wild-type TRIM5rh, TRIM5rh with the TRIM4 RING or B-box 2 domain (4R and 4B, respectively), and TRIM4 with the TRIM5rh RING or B-box 2 domains (5R and 5B, respectively). C, coprecipita- tion of wild-type TRIM4hu or TRIM4hu with the TRIM5rh RING or B-box 2 domain (5R and 5B, respectively) and TRIM5 with the TRIM4 RING or B-box 2 domain (4R and 4B, respectively). The co-IP indices are indicated below the Western blots.

Article Snippet: Western blotting was done with a polyclonal anti-TRIM5 antibody (Imgenex) and an anti- -actin antibody as control.

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

doi: 10.1074/jbc.m111.260406

Figure Lengend Snippet: FIGURE 8. Higher order associations of the TRIM5rh/TRIM4hu chimeras with replacements of both the RING and the B-box 2 domains. A, sche- maticdiagramofwild-typeTRIM5rhandTRIM4huandthechimeras.B,higher order association of the RING-B-box 2 (RB) chimeras, the B30.2(SPRY) domain chimera (4–5SPRY), and the wild-type parental TRIM5 and TRIM4 proteins. The 4RB chimera is a TRIM5rh protein in which both the RING and B-box 2 domains have been replaced by those of TRIM4. The 5RB chimera is a TRIM4 protein in which both the RING and B-box 2 domains have been replaced by those of TRIM5. The 4–5SPRY chimera is a TRIM4 protein with the B30.2(SPRY) domain replaced by that of TRIM5. The co-IP indices of the chimeras were normalizedtothoseassociatedwithTRIM4self-associationand/orTRIM5self- association and are indicated below the Western blots.

Article Snippet: Western blotting was done with a polyclonal anti-TRIM5 antibody (Imgenex) and an anti- -actin antibody as control.

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: mBio

Article Title: Spatiotemporal binding of cyclophilin A and CPSF6 to capsid regulates HIV-1 nuclear entry and integration

doi: 10.1128/mbio.00169-25

Figure Lengend Snippet: Increased CypA binding to HIV-1 capsid inhibits infection in a CPSF6-dependent manner. ( A ) OMK cells were infected with different amounts of WT HIV-1 and CA mutants (0.1–100 ng p24). Average luciferase values are shown for two independent experiments. ( B–E ) Infectivity of WT and mutant HIV-1 (10 ng p24) was determined after 48 h by luciferase activity in DMSO or 5–10 μM CsA in HeLa ( n = 3) ( B ), Jurkat ( n = 3) ( C ), primary CD4+ T cells ( n = 2) ( D ), and Jurkat PPIA −/ − ( n = 3) ( E ). ( F ) Primary CD4+ T cells were transduced with lentiviruses expressing control or TRIM5α miRNA prior to infection with WT and mutant HIV-1 (10 ng p24) in DMSO or 10 μM CsA. Infections were determined after 48 h by luciferase activity ( n = 2). Error bars represent standard errors of the mean (SEM). Comparisons between infection conditions were analyzed by unpaired t tests. P values of <0.05 were considered significant and significant values are denoted as *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. ns, P > 0.05.

Article Snippet: Lentiviral vectors encoding control or TRIM5α miRNAs (pAPM-D4-miR30-L1221 or pAPM-D4-miR30-TRIM5α; Addgene) were produced by co-transfection with the packaging plasmid pcHelp ( ) and pCMV-VSV-G ( ).

Techniques: Binding Assay, Infection, Luciferase, Mutagenesis, Activity Assay, Transduction, Expressing, Control