Journal: Journal of Biomedical Science

Article Title: Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions

doi: 10.1186/s12929-023-00990-8

Figure Lengend Snippet: UBA1 conveys Rab34 action on FABP5 stability to regulate lipid metabolism. A Transfected 3T3-L1 cells with the indicated plasmids (GFP-Rab34 or FABP5-c-Myc) were treated with MG132 (10 μmol/L, 12 h) and lysed under denaturing conditions. c-Myc-tagged FABP5 was purified by anti-c-Myc immunoprecipitation and ubiquitinated FABP5 was detected by Western Blot. An expression vector coding for hemagglutinin (HA)-tagged ubiquitin (HA-Ubiquitin) was employed for cotransfection of cells expressing FABP5-c-Myc, alone or in combination with GFP-Rab34. The graph shows the ratio of Ubiquitinated-FABP5-c-Myc immunosignal to Ponceau S immunosignal. Data are referred to values in non-transfected cells (100%) and expressed as mean ± SEM (n = 3 biological replicates). **P < 0.01; ***P < 0.001. B Co-immunoprecipitation analysis in cells expressing GFP-Rab34 and vectors coding for LD-associated proteins related to ubiquitination/deubiquitination processes: UBA1-c-Myc (top panel), UCHL3-c-Myc (middle panel), or ISG15-c-Myc (bottom panel). In each experimental setting, proteins were purified by anti-c-Myc immunoprecipitation and detected by Western Blot using anti-c-Myc or anti-GFP antibodies. C Representative immunoblots and quantification of FABP5 levels in 3T3-L1 cells transfected with GFP-Rab34 (+ , 0.8 µg/µL), UBA1-c-Myc (+ , 0.8 µg/µL; + + , 1.6 µg/µL) or both expression vectors in the absence or presence of MG132 (10 µmol/L, 12 h). Data represent the ratio of FABP5 immunosignal to β-actin immunosignal and referred to values in non-transfected cells (100%). Data are expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001 vs. non-transfected cells. $$ P < 0.01; $$$ P < 0.001 vs. their respective condition treated with MG132. # P < 0.05; ## P < 0.01. D, E Rescue experiments of FABP5 in 3T3-L1 cells expressing GFP-Rab34 and UBA1 siRNA (siUBA1), alone or in combination. At the end of the experiments, cells were processed for immunoblotting studies ( D ) (see also Fig. S4) and for measurement of TGs (lipogenesis) and glycerol content (lipolysis) ( E ). Data are referred to values in control cells (100%; Scr), and expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Plasmids coding for Ubiquitin C-Terminal Hydrolase L3 (UCHL3) (pCMV6-UCHL3-c-Myc) and ISG15 Ubiquitin Like Modifier (ISG15) (pCMV6-ISG15-c-Myc) were purchased from OriGene (Rockville, USA).

Techniques: Transfection, Purification, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Ubiquitin Proteomics, Cotransfection, Control

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. The mRNA levels of ISG15 and ISG56 genes on lesion and adjacent non-lesion skin samples in vivo . Holstein cattle were intravenously injected with LSDV for 14 days, and then lesion and non-lesion skin samples from the same cattle were dissected for RT-qPCR analysis of mRNA levels of ISG15 and ISG56 genes. B. Effects of LSDV on IFN-β-induced transcription of ISGs. MDBK cells (5 × 10 5 ) were left un-infected or infected with wild-type LSDV (MOI = 1) for 12 hours and then treated with IFN-β (100 ng/ml, final concentration) for 6 hours followed by RT-qPCR analysis of mRNA levels of the indicated genes. C. Effects of LSDV on IFN-β-induced phosphorylation of STAT1 and STAT2. MDBK (5 × 10 5 ) cells were left uninfected or infected with wild-type LSDV (MOI = 1) for 12 hours and then treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. Data shown in A-B are mean ± SD (n = 11 in A and n = 3 in B) from one representative experiment. These experiments were repeated at least twice with similar results. ns nonsignificant, **P < 0.01 (unpaired t-test).

Article Snippet: At 5 and 10 hours post-infection, orbital blood samples were collected, and the levels of Isg15 and Cxcl10 in the serum were measured using ELISA kits (CSB-EL011843MO, CUSABIO; EK0736, BOSTER).

Techniques: In Vivo, Injection, Quantitative RT-PCR, Infection, Concentration Assay, Phospho-proteomics, Western Blot

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. Effects of LSDV122-deficiency on LSDV-induced production of serum ISG15 and CXCL10. Seven-week-old C57BL/6 mice were intravenously injected with medium (mock, n = 4), LSDV (2.6 × 10 5 pfu each mouse, n = 6) or LSDVΔ122 (2.6 × 10 5 pfu each mouse, n = 5). The orbital blood of viral infected mice was collected at 5 and 10 hours post infection, and the orbital blood of mock injected mice was collected at 5 hours post injection for ELISA analysis of CXCL10 and ISG15 levels. B-D. Effects of LSDV122-deficiency on LSDV-induced transcription of antiviral genes in different tissues of mice. Seven-week-old C57BL/6 mice were intravenously injected with medium (mock, n = 4), LSDV (2.6 × 10 5 pfu each mouse, n = 6) or LSDVΔ122 (2.6 × 10 5 pfu each mouse, n = 5). Spleen (B), liver (C) and lung (D) of mice were collected at 5 or 10 hours post viral infection or 5 hours post mock injection for RT-qPCR analysis of mRNA levels of the indicated genes. Data shown in A-D are mean ± SD. These experiments were repeated at least twice with similar results. *P < 0.05; **P < 0.01 (unpaired t-test).

Article Snippet: At 5 and 10 hours post-infection, orbital blood samples were collected, and the levels of Isg15 and Cxcl10 in the serum were measured using ELISA kits (CSB-EL011843MO, CUSABIO; EK0736, BOSTER).

Techniques: Injection, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Molecular Immunology

Article Title: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase

doi: 10.1016/j.molimm.2009.03.030

Figure Lengend Snippet: Renal C3 stain in C fh −/− mice 24 h after the injection of purified mCFH. Kidney sections were stained for C3 using polyclonal anti-mouse C3. Linear capillary wall staining is evident in C fh −/− mice injected with PBS or 0.75 μg of LPS. In contrast, mesangial staining is seen in C fh −/− mice 24 h after injection of 1 mg of mCFH. C3 staining is present along Bowman's capsule and within tubulo-interstitium of wild-type mice. Tubulo-interstitial staining is absent in C fh −/− mice injected with PBS or LPS. However, after administration of mCFH, tubulo-interstial staining is now evident in the C fh −/− animals. Original magnification 40×. WT: wild-type.

Article Snippet: After each chromatography step the samples were checked for the presence of CFH by western blot using polyclonal cross-reactive anti-human CFH (Quidel, CA, USA).

Techniques: Staining, Injection, Purification

Journal: Molecular Immunology

Article Title: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase

doi: 10.1016/j.molimm.2009.03.030

Figure Lengend Snippet: Nature of C3 bound to the glomeruli 24 h after the administration of mCFH. (A) Kidney sections were stained for C3 using polyclonal anti-mouse C3d (red stain) or polyclonal anti-mouse C3 (green stain) antibodies. In the mice injected with PBS linear capillary wall staining was evident using both antibodies. However, in the C fh −/− mice injected with mCFH a mesangial staining pattern was evident using anti-mouse C3 antibody whilst the linear capillary wall staining pattern remained unchanged with the anti-C3d antibody. The merged images showed that the areas of mesangial reactivity did not co-stain with the anti-C3d. Original magnification 40×. (B) Western blot analysis of C3 under reducing conditions using solubilised laser dissected glomerular tissue from C fi −/− , C fh −/− or C fh −/− mice injected with mCFH. To demonstrate the positions of the intact C3 α -chain, the α ′ chain of C3b and the β -chain of C3 plasma from both wild-type (intact C3) and C fi −/− mice (in which all C3 is circulating as C3b) was also run on the gel. C3 α -chain fragments and the C3 β -chain were evident in C fh −/− mice with or without the administration of mCFH. In contrast no α -chain fragments were evident in glomeruli from C fi −/− animals. (C) Glomerular C3 staining in mice with combined deficiency of CFH and CFI (C fh −/− .C fi −/− ) that have been given CFI. Following adminstration of sera containing CFI, linear capillary wall C3 staining develops that is reactive with both the anti-C3 and anti-C3d antibody. Original magnification 40×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Article Snippet: After each chromatography step the samples were checked for the presence of CFH by western blot using polyclonal cross-reactive anti-human CFH (Quidel, CA, USA).

Techniques: Staining, Injection, Western Blot, Clinical Proteomics

Journal: Molecular Immunology

Article Title: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase

doi: 10.1016/j.molimm.2009.03.030

Figure Lengend Snippet: Detection of CFH-related proteins in C fh −/− mice and tracking mCFH after administration to C fh −/− mice. (A) Serum western blot for CFH using cross-reactive polyclonal anti-human CFH antibody. (B) Renal sections from C fh −/− mice 2 h after the injection of Alexa-488-tagged CFH. At this time point the Alexa-488-tagged CFH was detected within the mesangium and tubulointerstitium but not along the GBM. At this time-point linear C3 staining is evident in C fh −/− mice similar to unmanipulated C fh −/− mice (far right panel). (C) Kidney sections immunostained for CFH using anti-human CFH antibody from wild-type, unmanipulated C fh −/− mice and C fh −/− mice 24 h after injection of mCFH. Some mesangial reactivity is present in wild-type glomeruli (left panel) whilst marked linear capillary wall staining is evident in the unmanipulated C fh −/− mice (middle panel). In contrast, a mesangial staining pattern was evident in C fh −/− mice 24 h after injection of mCFH (right panel) with very little staining along the capillary walls (arrows). Original magnification, 40×.

Article Snippet: After each chromatography step the samples were checked for the presence of CFH by western blot using polyclonal cross-reactive anti-human CFH (Quidel, CA, USA).

Techniques: Western Blot, Injection, Staining

Journal: Molecular Immunology

Article Title: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase

doi: 10.1016/j.molimm.2009.03.030

Figure Lengend Snippet: Influx of neutrophils into the glomeruli after the administration of mCFH. (A) Glomerular neutrophil numbers in C fh −/− mice 24 h after injection of PBS, 0.75 μg of LPS or 1 mg of mCFH. Bars denote median values with standard deviation (B) Glomerular neutrophils evident in representative glomerular light microscopic images in mice that had received mCFH but not LPS. Original magnification 40× (C) FACS detection of blood neutrophils using rat IgG2b anti-mouse GR-1 antibody in Cfh −/− mice before, 24 and 48 h after neutrophil depletion. Neutrophil depletion was achieved by a single injection of rat monoclonal IgG2b anti-murine neutrophil Ly.6G (αNE). (D) Glomerular neutrophil numbers in neutrophil-depleted C fh −/− mice 24 h after injection of mCFH demonstrating absence of glomerular neutrophils in mice pre-treated with the αNE antibody. (E) Glomerular C3 stain using polyclonal anti-mouse C3 antibody in neutrophil-depleted mice treated with mCFH. The absence of neutrophils did not influence the change in glomerular C3 staining seen after the administration of mCFH. Compare this image with that shown in . Original magnification 40×. WT: wild-type.

Article Snippet: After each chromatography step the samples were checked for the presence of CFH by western blot using polyclonal cross-reactive anti-human CFH (Quidel, CA, USA).

Techniques: Injection, Standard Deviation, Staining