Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 knockdown increases HIV-1 replication. a Jurkat T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1 or siABIN1-2) or scramble siRNA (siNC) as described in “ ”. 36 h post transfection (hpt), cells were counted and challenged with HIV-1 strain NL4-3. At 1, 3, 5 days post infection, the cells and supernatants were harvested to measure viral production by p24 ELISA of the supernatants, against a standard curve. Cells were lysed and subjected to Western Blots to determine the knockdown efficiency of ABIN1. The lower panel showed the ABIN1 knockdown efficiency at day 5 as the representative. b The experiments were conducted similarly as in ( a ), except that human primary CD4+ T lymphocytes were used. c , d Determination of ABIN1 mRNA level after viral infection. Jurkat cells were challenged with HIV(NL4-3) as in a , b , at 0, 24, 72 hpi, cells were harvested for RNA extraction. The mRNA levels of ABIN1 and HIV-1 gag was measured by real time PCR, normalized to cellular GAPDH. Data are shown as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC transfected cells or samples collected at 0 hpi. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Knockdown, Transfection, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, RNA Extraction, Real-time Polymerase Chain Reaction

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 depletion stimulates the activity of HIV-1 promoter (Ghost-CXCR4 (X4) cells). a – e Ghost-X4 cells stably transfected a HIV-1 LTR driving GFP expression were treated with siNC, siABIN1-1, or siABIN1-2 before challenged with HIV-1(NL4-3). 48 hpi, cells were harvested and subjected to flowcytometry analysis. The effect of ABIN1 depletion on HIV-1 replication was determined by calculating the percentage of GFP positive cells, the relative mean fluorescence intensity (MFI) and relative median of GFP intensity of these GFP positive cells, over 10,000 cells were measured for each sample. a Indicates the basal level of GFP in non-infected Ghost-X4 cells. b – d Comparison of the cell counts at different GFP intensity after viral infection following ABIN1 knockdown. e The emphasis of the comparison. f , g Changing fold of MFI ( f ) and GFP intensity median ( g ) in infected cells following transfection of siRNAs targeting ABIN1 relative to siNC. The basal MFI and median of GFP intensity in siNC transfected cells were both set to 1000. h Representative of ABIN1 knockdown efficiency in Ghost-X4 cells. Data are shown as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC indicated. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Activity Assay, Stable Transfection, Transfection, Expressing, Fluorescence, Infection, Comparison, Knockdown

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 depletion stimulates the activity of HIV-1 promoter (HeLa cells). a – e HeLa cells were transfected with siNC, siABIN1-1, or siABIN1-2 for 36 h, then cells were challenged with VSV-G pseudotyped luciferase reporter HIV-1(Luc). Cells were harvested at the indicated time points post infection for luciferase activity analysis, DNA or RNA extraction. a Luciferase activities of the infected cells were monitored at 48 hpi ( upper panel ) and ABIN1 knockdown efficiency was determined by Western Blots as the representative ( lower panel ). b – d Total DNA of the infected cells were extracted and quantified for subsequent real-time PCR at b 12 hpi for late RT levels, c 24 hpi 2-LTR-circle levels, d 48 hpi proviral DNA levels. e Total RNA was extracted at 48 hpi, and used for quantitation of HIV-1 mRNA. f The effect of overexpressed ABIN1 on LTR promoter activity were determined by luciferase assays after co-transfection of Flag-ABIN1 expressing plasmid or vector control together with pNL4-3.Luc.R-.E-, and pRL-TK in HEK-293T cells for 24 h. g Expression of Flag-ABIN1 was determined by Western Blots. The real-time PCR analysis of DNA and RNA extract were normalized to cellular β-globin and GAPDH, respectively. The primer pairs used were as described in “ ”. Data are represented as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC or NC indicated. * p < 0.05; ** p < 0.01; *** p < 0.001, ns not significant

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Activity Assay, Transfection, Luciferase, Infection, RNA Extraction, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Quantitation Assay, Cotransfection, Expressing, Plasmid Preparation, Control

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 suppresses HIV-1 Tat ubiquitination via its ubiquitin binding property. a HEK-293T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1, siABIN1-2) or siNC for 24 h, then a second round transfection of Flag-Tat expressing constructs was performed for another 24 h, and cells were then harvested and assayed as described in “ ”. b After transfection with Myc-ABIN1 and Flag-Tat expressing plasmids for 24 h as described in “ ”, the ubiquitination of Tat in HEK-293T cells was analyzed by Flag IP under denaturing conditions as in ( a ). c HEK-293T cells were co-transfected with plasmids encoding Myc tagged wild-type ABIN1 or ABIN1-QE2 mutant and Flag-Tat for 24 h, cells were then harvested and analyzed as in ( a ). d The effect of overexpressed ABIN1 and QE2 mutant were determined by luciferase assays after HIV-1(Luc) infection following ABIN1, QE2 or vector control (NC) transfection in HeLa cells. The panel below indicates the expression of ABIN1 or its mutant. β-actin was detected as sample loading control. Data are represented as mean ± SD of triplicate samples, all data and Western Blots are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to NC indicated. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Ubiquitin Proteomics, Binding Assay, Transfection, Expressing, Construct, Mutagenesis, Luciferase, Infection, Plasmid Preparation, Control, Western Blot

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 negatively regulates the K63-linked polyubiquitination of HIV-1 Tat. a To determine the effect of ABIN1 on the stability of Tat, HEK-293T cells were transfected with Myc-ABIN1 and Flag-Tat encoding plasmids as indicated for 24 h, followed by treatment of CHX at the final concentration of 10 μg/ml for 6 h to arrest cell translation. The levels of Tat protein were detected by Western Blot. b To elucidate whether ABIN1 targeting Tat for proteasome-dependent degradation, HEK-293T cells were transfected with siABIN1-1 or siNC for 24 h, followed by a second round transfection of Flag-Tat encoding plasmids, cells were then treated with MG-132 at the final concentration of 10 ng/ml for 2 h to block proteasomal degradation. The accumulation of Flag-Tat were detected by IB. c The type of ubiquitination regulated by ABIN1 was explored in HEK-293T or HEK-293T ABIN1-KO cells by immunoprecipitation as in Fig. a after transfected with Flag-Tat, together with wild-type HA–Ub, HA–Ub K63R or HA–Ub K48R mutant expression vectors for 24 h. β-actin was detected as sample loading control. The blots were representatives of at least three independent experiments achieving similar results

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Transfection, Concentration Assay, Western Blot, Blocking Assay, Ubiquitin Proteomics, Immunoprecipitation, Mutagenesis, Expressing, Control

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 specifically decreases the ubiquitination of Tat at Lys71. a HEK-293T cells were treated with siRNAs and plasmids similar as in Fig. a as indicated, except that Flag-Tat-K71R mutant was used to assess the ubiquitination of wild-type Tat and Tat K71R mutant in the presence or absence of ABIN1. b HeLa cells were infected with HIV-1(Luc) or HIV-1(Luc-K71R) at the same MOI of 0.2, replication activity (Repl. activity) was assessed by luciferase assays 48 hpi to evaluate the importance of Lys71 in Tat. c , d HEK-293T cells were treated with siRNAs targeting ABIN1 (siABIN1-1, siABIN1-2) or siNC ( c ) or transfected with wild-type ABIN1 or QE2 mutant encoding vectors and challenged with HIV-1(Luc) or HIV-1(Luc-K71R) ( d ), then subjected to luciferase assays 24 hpi to monitor the replication efficiency, and the cell lysates were collected and cleared for detection of knockdown efficiency and overexpression level of ABIN1/mutant. The blots are representatives of at least three independent experiments achieving similar results

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Ubiquitin Proteomics, Mutagenesis, Infection, Activity Assay, Luciferase, Transfection, Knockdown, Over Expression

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 regulates the ubiquitination of Tat by modulating the distribution of HDM2. a HEK-293T ABIN1-KO cells were transfected with plasmids encoding Myc-ABIN1 or Myc-QE2, together with Myc-HDM2, Flag-Tat or plasmid vectors as indicated, and subjected to IP as in Fig. a. b HeLa cells were transfected with expression vectors encoding or Myc-ABIN1, Myc-QE2, Myc-HDM2, Flag-HDM2 or Flag-Tat individually ( upper panel ) or in combination ( middle and lower panel ) as indicated for 24 h. Then cells were washed with PBS and harvested for Immunofluorescence analysis. Mouse anti-Myc and rabbit anti-Flag antibodies were used as primary antibodies, FITC-conjugated goat anti-rabbit IgG and Rhodamine-conjugated goat anti-mouse IgG were used to detect the two proteins. The consensus scale bar was 20 μm

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Immunofluorescence

Journal: iScience

Article Title: Muscle glycome in idiopathic inflammatory myopathies: Impact in IL-6 production and disease prognosis

doi: 10.1016/j.isci.2023.107172

Figure Lengend Snippet:

Article Snippet: Polyclonal DC-SIGN rabbit IgG , Biorad , Cat#AHP627; RRID: AB_323683.

Techniques: Plasmid Preparation, Recombinant, Reverse Transcription, Random Hexamer, Protease Inhibitor, Staining, Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 1 OTUD4 is downregulated in the lung tissue of COPD patients and CSE-induced emphysema mouse model. A Representative result of IHC (400× magnification) for OTUD4 in human lung tissue. B Western blotting to detect OTUD4 protein expression in human lung tissue. C OTUD4 was detected with IHC in the lung tissue of mice model. D Western blotting was applied to detect the OTUD4 protein expression in the lung tissue of mice. Data represents as mean ± SD of three independent experiments. *P < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Western Blot, Expressing

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 2 OTUD4 deficiency accelerates CSE induced emphysema via increasing airway epithelium apoptosis. A Representative HE staining images (200×) of lung tissue were from control mice and OTUD4 deficiency mice. DI (%) and MLI (μm) were measured. Immunoblotting (B) and IHC (C) were used to detect OTUD4 protein expression (400×). D Apoptosis of lung cells in indicated group of mice were detected via TUNEL assay (400×). Apoptosis index (apoptotic cells/ total cells, %) was calculated. E BCL2, Bax and cleaved-caspase 3 protein expression was detected via immunoblotting. Lenti-NC: negative control; Lenti-shOTUD4: OTUD4 knockdown. Data were shown as mean ± SD of three independent experiments. *P < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Staining, Control, Western Blot, Expressing, TUNEL Assay, Negative Control, Knockdown

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 3 Enhanced OTUD4 alleviates emphysema by reducing cell apoptosis. A Western blot to detect the expression of OTUD4 protein in the lung tissue of mice. B Representative results of IHC (400×) for OTUD4 in different groups were shown. C Histological changes of lung sections were shown with H&E (200×) staining. Morphometric measurements of MLI (μm) and DI (%) were plotted. D The expression of apoptotic protein including BCL2, Bax and cleaved caspase 3 were detected in the lung tissue of mice. E Apoptotic nuclei were detected by TUNEL staining (400×) in the lung tissue of mice. Lenti-EV: empty vector, Lenti-OTUD4: OTUD4 overexpression; Data were shown as mean ± SD of three independent experiments. *P < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Western Blot, Expressing, Staining, TUNEL Assay, Plasmid Preparation, Over Expression

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 4 OTUD4 decreases in CSE induced lung epithelial cells. A–D OTUD4 relative expression was assayed by western blotting in type II epithelial A549 cells, HBEs and BEAS-2b. E RT-qPCR to detect the mRNA level of OTUD4 in CSE treated BEAS-2b cells. Data represents as mean ± SD of three independent experiments. *P < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 5 OTUD4 regulates apoptosis in lung epithelial cells induced by CSE. A, B The mRNA and protein level of OTUD4 were confirmed with RT-qPCR and western blot. C OTUD4, BCL2, Bax, cleaved-caspase 3 or β-actin were assayed with western blotting after OTUD4 knockdown. D TUNEL staining (400×) to detect apoptotic nuclei of BEAS-2b cells. E Western blot to detect the protein level of BCL2, Bax and cleaved caspase 3. F Apoptotic nuclei of BEAS-2b cells were detected by TUNEL staining (400×). Data were shown as mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, TUNEL Assay, Staining

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 6 OTUD4 deubiquitinates PAI-1 and inhibits its proteasomal degradation. A Western-blot assayed the protein level of PAI-1 in BEAS-2b cells. B PAI-1 protein expression in the lung tissue of mice was detected by western blotting. C CHX or MG132 was subjected to BEAS-2b cells. PAI-1 is with a half-life of about 3.3 h. D, E PAI-1 protein expression was detected by western blotting in BEAS-2b cells after OTUD4 knockdown or overexpression with CHX treatment. Data represents as mean ± SD of three independent experiments. *P < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Western Blot, Expressing, Knockdown, Over Expression

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 7 OTUD4 deubiquitinates PAI-1 and inhibits its proteasomal degradation. A, B BEAS-2b cells were treated with CSE or MG132, CSE or MG132 was subjected to BEAS-2b cells after OTUD4 knockdown, Western blotting was used to detect OTUD4 and PAI-1 protein level. C Immunofluorescence stanning (400×) of OTUD4 (green), PAI-1 (red) and DAPI (blue) and merged image (upper panel). D Immunoprecipitation of OTUD4 and PAI-1 (lower panel). E Ubiquitination assay of PAI-1 in CSE induced BEAS-2b cells after OTUD4 overexpression. F WT, K48, or K63 Ub was co-transfected with PAI-1 and OTUD4 into HEK293T cells. After treatment with 5 μM MG132 for 5 h, cell lysates were subjected to ubiquitination assay. Data represents as mean ± SD of three independent experiments. *P < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Knockdown, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Over Expression, Transfection

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 8 Inhibition of PAI-1 perturbs the protective effect of OTUD4 on CSE induced apoptosis in BEAS-2b cells. A OTUD4 overexpressed cells were treated with PAI-1 inhibitor (tiplaxitinin) or CSE. OTUD4, BCL2, Bax, and Cleaved-caspase3 were detected by western blot. B Densitometry of OTUD4, BCL2, Bax, Cleaved-caspase3. C Apoptotic nuclei were detected by TUNEL staining (400×) in OTUD4 overexpressed BEAS-2b cells treated with CSE or tiplaxitinin. D Apoptotic index (%) was plotted. Data represents as mean ± SD of three independent experiments. *P < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Inhibition, Western Blot, TUNEL Assay, Staining

Journal: Cell death & disease

Article Title: CSE reduces OTUD4 triggering lung epithelial cell apoptosis via PAI-1 degradation.

doi: 10.1038/s41419-023-06131-1

Figure Lengend Snippet: Fig. 9 Inhibition of PAI-1 perturbs the protective effect of OTUD4 on CSE induced apoptosis in lung tissue of mice. A, B Histological changes of lung sections were shown with H&E (200×) staining. Morphometric measurements of MLI (μm) and DI (%) were plotted. C-D Representative results of IHC (400×) for OTUD4 in different groups were shown. E, F Western blot to detect the expression of OTUD4, BCL2, Bax and cleaved caspase 3 protein in the lung tissue of mice. G, H Apoptotic nuclei were detected by TUNEL staining (400×) in the lung tissue of mice. Lenti-EV: empty vector, Lenti-OTUD4: OTUD4 overexpression; Data were shown as mean ± SD of three independent experiments. *P < 0.05.

Article Snippet: OTUD4 antibody (Cat: #A304–605A) was form Bethyl company (Montgomery, USA), PAI-1(Cat: sc-5297) was from Santa Cruz Biotechnology (Shanghai, China), Bax antibody (Cat: 50599-2-Ig) was from proteintech (Rosemont, USA).

Techniques: Inhibition, Staining, Western Blot, Expressing, TUNEL Assay, Plasmid Preparation, Over Expression

Journal: Frontiers in Veterinary Science

Article Title: Unveiling the role of hexon-associated host proteins in fowl adenovirus serotype 4 replication

doi: 10.3389/fvets.2025.1562872

Figure Lengend Snippet: Identification of the interaction of the LMH cell proteins with the FAdV-4 hexon. Immunoprecipitation and western bolt were conducted to confirm the expression of bait and prey proteins in LMH cells (A–C) . Co-immunoprecipitated of hexon protein (113 kDa) with CCT5 (60 kDa), CCT7 (59 kDa), HSP70 (68 kDa). The cell lysates of the two groups (flag-TST-hexon and flag control vector) were incubated with anti-flag magnetic beads at 4°C overnight and then eluted with flag eluent to obtain Co-IP products. Co-IP products were boiled in loading buffer, and then the flag labeled CCT5, CCT7, HSP70 antibodies, or hexon specific antibodies were used for western bolt detection.

Article Snippet: Antibodies specific for CCT5 (catalog no. 11603-1-AP), CCT7 (catalog no. 15994-1-AP), and HSP70 (catalog no. 10995-1-AP) were procured from Proteintech, China.

Techniques: Immunoprecipitation, Western Blot, Expressing, Control, Plasmid Preparation, Incubation, Magnetic Beads, Co-Immunoprecipitation Assay, Labeling

Journal: Nature Communications

Article Title: Destabilized near-infrared fluorescent nanobodies enable background-free targeting of GFP-based biosensors for imaging and manipulation

doi: 10.1038/s41467-024-51857-x

Figure Lengend Snippet: a Design of NIR Nb for GFP (Nb GFP ) (PDB ID 3OGO) with indicated position for insertion of miRFP718nano (PDB ID 7LSD). CDRs of Nb GFP are highlighted with violet and the position of miRFP718nano insertion is indicated in red. b Co-expression of NIR Nb GFP containing inserted miRFP718nano (NIR-Fb (718) ) with cognate mEGFP antigen in the cytoplasm (upper panels) or in the nucleus (bottom panels) of HeLa cells. c Upper panels, fluorescent images of HeLa cells co-expressing near-infrared Nb for p24 HIV protein with inserted miRFP670nano3 fluorescent protein (NIR-Fb 59H10 ) and cognate antigen, NES-p24-mTagBFP2. Middle panels, fluorescent images of HeLa cells co-expressing NIR Nb for GFP with inserted miRFPnano718 (NIR-Fb (718) ) and EGFP-H2B. Bottom panels, fluorescent images of HeLa cells co-expressing NIR-Fb 59H10 and NIR-Fb (718) and two respective antigens, NES-p24-mTagBFP2 and EGFP-H2B. Patterns of co-expression are indicated with yellow and white arrows, respectively. For imaging of mTagBFP2 and mEGFP, 390/40 nm excitation and 460/40 nm emission and 480/40 nm excitation and 530/40 nm emission filters we used, respectively. For imaging of NIR-Fb 59H10 and NIR-Fb (718) , 605/40 excitation and 667/30 emission, and 682/12 excitation and 721/42 emission filters were used, respectively. Scale bar, 40 μm. For more details, see “Statistics and reproducibility” section of the Methods.

Article Snippet: To generate NES-p24-mEGFP plasmid, a fusion of HIV p24 with mEGFP was amplified and inserted into the pcDNA plasmid with an NES signal (Addgene no.164052) at the BamHI/XhoI sites.

Techniques: Expressing, Imaging