Journal: Molecular Vision

Article Title: Effect of curcumin on the modulation of αA- and αB-crystallin and heat shock protein 70 in selenium-induced cataractogenesis in Wistar rat pups

doi:

Figure Lengend Snippet: Effects of curcumin on immunohistochemistry of αA- and αB-crystallin in the eye lens of Wistar rat pups exposed to selenium. A : This image shows the control lens incubated in saline alone without any antibody treatment. B : This is a control lens that was incubated with saline and subjected to antibody treatment. C : Lens from rat pups administered with selenium alone. D : Lens from rat pups administered with selenium and curcumin simultaneously. E : Lens from rat pups administered with selenium first and then treated with curcumin after 24 h. F : Lens from rat pups pretreated with curcumin and then administered with selenium after 24 h. Lens sections were preincubated with αA- and αB-crystallin polyclonal antirabbit Immunoglobulin G (IgG) antibody (1:3,000 dilution) and subsequently with goat anti-rabbit IgG-horse radish peroxidase (HRP) conjugate (1:3,000 dilution). The immunoreactivity was developed with 0.01% 3,3-diaminobenzidine tetrahydrochloride (DAB) and H 2 O 2 . Note the brown color formation indicative of peroxidase reaction in the nucleus. Image G , negative control, lens treated with goat anti-rabbit IgG-HRP and developed using DAB and hydrogen peroxide (H 2 O 2 ). The figure shows the high level of αA- and αB-crystallin expression induced by selenium-mediated oxidative stress. This increased crystallin expression and aggregate formation was prevented by curcumin pretreatment.

Article Snippet: After washing with TBS containing 0.05% Tween 20, the sections were incubated with the primary antibody, αA- and αB-crystallin polyclonal anti-rabbit IgG antibody (BD Biosciences, San Jose, CA), at a dilution of 1:500 overnight at 4 °C.

Techniques: Immunohistochemistry, Incubation, Negative Control, Expressing

Journal: Molecular Vision

Article Title: Effect of curcumin on the modulation of αA- and αB-crystallin and heat shock protein 70 in selenium-induced cataractogenesis in Wistar rat pups

doi:

Figure Lengend Snippet: Immunoblot expression of αA- and αB-crystallin in control and experimental group of animals. Lane I, eye lens protein from control (physiologic saline) rat pups (group I); lane II, eye lens protein from selenium-injected rat pups (group II); lane III, eye lens protein from rat pups administered selenium and curcumin simultaneously (group III); lane IV, eye lens protein from rat pups injected with selenium 24 h before being administered with curcumin (group IV); and lane V, eye lens protein from rat pups administered with curcumin 24 h before being injected with selenium (group V). The separated lens protein was preincubated with αA- and αB-crystallin polyclonal antirabbit IgG antibody (1:3,000 dilution) and subsequently with goat antirabbit IgG-HRP (1:3,000 dilution). The immunoreactivity was developed with 0.01% DAB and H 2 O 2 . β-Actin refers to house keeping protein expression and its levels are constant across all treatment groups indicating the normal behaviour of lenses under various treatment. The figure clearly shows increased αA- and αB-crystallin protein expression under selenium-mediated oxidative stress. This increased crystallin protein expression was prevented by curcumin pretreatment.

Article Snippet: After washing with TBS containing 0.05% Tween 20, the sections were incubated with the primary antibody, αA- and αB-crystallin polyclonal anti-rabbit IgG antibody (BD Biosciences, San Jose, CA), at a dilution of 1:500 overnight at 4 °C.

Techniques: Western Blot, Expressing, Injection

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Frontiers in Pharmacology

Article Title: The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling

doi: 10.3389/fphar.2022.816551

Figure Lengend Snippet: PINK1/Parkin-dependent mitophagy induces apoptosis during mitochondrial stress (A) . Western blot analysis of mitophagy proteins and the pro-apoptosis signal in Hela (no Parkin) cells and Hela Parkin cells after the AO (Antimycin A+ oligomycin) treatment. Cells were treated with AO at 5 ug/ml for 0, 3, 6, and 9 h. Beta Actin served as the loading control. These are representative figures from three independent experiments (B) . Cell viability assay of Hela (no Parkin) cells and Hela Parkin cells after AO treatment. Cells were treated with AO at 5 ug/ml for 24 h and then incubated with resazurin for 2 h. Fluorescence was read using 544 nm excitation and 590 nm emission wavelength. It is a representative figure from three independent experiments (C) . Western blot analysis of mitophagy proteins and the pro-apoptosis signal in Hela Parkin cells and Hela Parkin with PINK1 KO cells after the AO treatment. Cells were treated with AO at 5 ug/ml for 0, 3, 6, and 9 h. Beta Actin served as the loading control (D) . Cell viability assay of Hela Parkin cells and Hela Parkin with USP30 overexpression cells after AO treatment with or without ST-539. Cells were treated with AO at 5 ug/ml w/o 10 ug/ml ST-539 for 24 h and then incubated with resazurin for 2 h. Fluorescence was read using544 nm excitation and 590 nm emission wavelength (E) . Western blot analysis of mitophagy proteins and the pro-apoptosis signal in Hela Parkin cells and Hela Parkin with USP30 overexpression cells after the AO treatment w/o ST-539. Cells were treated with AO at 5 ug/ml w/o 10 ug/ml ST-539 for 0, 3, and 6 h. Beta Actin served as the loading control (F) . Cell viability assay of Hela ATG5 knockout cells, Hela ATG5 knockout Parkin cells, and Hela ATG5 knockout Parkin USP30 cells after AO treatment. Cells were treated with AO at 5 ug/ml for 24 h and then incubated with resazurin for 2 h. Fluorescence was read using544 nm excitation and 590 nm emission wavelength.

Article Snippet: Primary antibodies used as described: USP30 (Sino Biological Inc., 14,548-RP01, 1:500); p -AKT (Cell Signaling Technology, 4060S, 1:1000); Parkin (Cell Signaling Technology, 4211S, 1:1000); AKT (Cell Signaling Technology, 4685S, 1:1000); Cleaved PARP (Cell Signaling Technology, 5625S, 1:1000); OPTN (Proteintech, 10837-I-AP, 1:1000); p -mTOR (Cell Signaling Technology, 5536S, 1:1000); mTOR (Cell Signaling Technology, 2983S, 1:1000); TOM20 (Cell Signaling Technology, 42406S, 1:1000); NDP52 (Cell Signaling Technology, 60732S, 1:1000); p-P70S6K (Cell Signaling Technology, 9234P, 1:1000); P70S6K (Cell Signaling Technology, 9202S, 1:1000); LC3A/B (Cell Signaling Technology, 4108S, 1:1000); Beta-Actin (Cell Signaling Technology, 3700S, 1:1000).

Techniques: Western Blot, Control, Viability Assay, Incubation, Fluorescence, Over Expression, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling

doi: 10.3389/fphar.2022.816551

Figure Lengend Snippet: USP30 upregulates AKT/mTOR signal (A) . Western blot analysis of AKT/mTOR pathway proteins in Hela Parkin cells and Hela Parkin with USP30 overexpression cells after the AO treatment. Cells were treated with AO at 5 ug/ml for 0, 3, 6, and 9 h. Beta Actin served as the loading control. 2 (B) . Western blot analysis of AKT signal in Hela Parkin cells and Hela Parkin with USP30 overexpression cells after the AO treatment w/o ST-539. Cells were treated with AO at 5 ug/ml w/o ST-539 for 0, 3, and 6 h. Beta Actin served as the loading control. 2 (C) . Western blot analysis of AKT and cleaved PARP in Hela Parkin and Hela Parkin USP30 cells after the AO treatment w/o chloroquine. Cells were treated with AO at 5 ug/ml for 0, 3, and 6 h with DMSO or 10 uM chloroquine to inhibit autophagy/mitophagy. Beta Actin served as the loading control. 2 (D) . Western blot analysis of AKT and cleaved PARP in Hela ATG5 knockout Parkin and Hela ATG5 knockout Parkin USP30 cells after the AO treatment. Cells were treated with AO at 5 ug/ml for 0, 3, and 6 h. Beta Actin served as the loading control.

Article Snippet: Primary antibodies used as described: USP30 (Sino Biological Inc., 14,548-RP01, 1:500); p -AKT (Cell Signaling Technology, 4060S, 1:1000); Parkin (Cell Signaling Technology, 4211S, 1:1000); AKT (Cell Signaling Technology, 4685S, 1:1000); Cleaved PARP (Cell Signaling Technology, 5625S, 1:1000); OPTN (Proteintech, 10837-I-AP, 1:1000); p -mTOR (Cell Signaling Technology, 5536S, 1:1000); mTOR (Cell Signaling Technology, 2983S, 1:1000); TOM20 (Cell Signaling Technology, 42406S, 1:1000); NDP52 (Cell Signaling Technology, 60732S, 1:1000); p-P70S6K (Cell Signaling Technology, 9234P, 1:1000); P70S6K (Cell Signaling Technology, 9202S, 1:1000); LC3A/B (Cell Signaling Technology, 4108S, 1:1000); Beta-Actin (Cell Signaling Technology, 3700S, 1:1000).

Techniques: Western Blot, Over Expression, Control, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling

doi: 10.3389/fphar.2022.816551

Figure Lengend Snippet: USP30 in cancer treatment. 3 (A) . Cell viability assay of Hela Parkin USP30 cells after AKT/mTOR inhibitors treatment w/o ST. Cells were treated with 10 uM MK2206 or 10uM Rapamycin or 1uM Torin1 for 48 h with DMSO or 10 ug/ml ST-539 and then incubated with resazurin for 2 h. Fluorescence was read using 544 nm excitation and 590 nm emission wavelength. 3 (B) . Cell viability assay on Jurkat T cells after 72 h MK2206 treatment with ST. Cells were treated with MK2206 and ST in the concentration gradient manner for 72 h. After the treatment, cells were incubated with resazurin for 2 h. Fluorescence was read using544 nm excitation and 590 nm emission wavelength. Each dot is the mean value of three biologically independent experiments. Trend lines are non-linear regression fitting curves. 3 (C) . Western blot analysis of AKT, mitophagy, and pro-apoptosis signal in Jurkat T cells treated with DMSO, MK2206, or ST. Cells were treated with DMSO, MK2206, ST, or MK220 + ST for 24 h. Beta Actin served as the loading control.

Article Snippet: Primary antibodies used as described: USP30 (Sino Biological Inc., 14,548-RP01, 1:500); p -AKT (Cell Signaling Technology, 4060S, 1:1000); Parkin (Cell Signaling Technology, 4211S, 1:1000); AKT (Cell Signaling Technology, 4685S, 1:1000); Cleaved PARP (Cell Signaling Technology, 5625S, 1:1000); OPTN (Proteintech, 10837-I-AP, 1:1000); p -mTOR (Cell Signaling Technology, 5536S, 1:1000); mTOR (Cell Signaling Technology, 2983S, 1:1000); TOM20 (Cell Signaling Technology, 42406S, 1:1000); NDP52 (Cell Signaling Technology, 60732S, 1:1000); p-P70S6K (Cell Signaling Technology, 9234P, 1:1000); P70S6K (Cell Signaling Technology, 9202S, 1:1000); LC3A/B (Cell Signaling Technology, 4108S, 1:1000); Beta-Actin (Cell Signaling Technology, 3700S, 1:1000).

Techniques: Viability Assay, Incubation, Fluorescence, Concentration Assay, Western Blot, Control

Journal: Frontiers in Endocrinology

Article Title: A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

doi: 10.3389/fendo.2024.1407859

Figure Lengend Snippet: co-expressed mRNA of NONMMUG018089.2.

Article Snippet: Primary antibodies are rabbit anti-mouse Col11a2 antibody (YT1009, 1:500, Immunoway, China) and mouse anti-mouse β-actin antibody (TA811000, 1:2000, OriGene, USA) while mouse and rabbit second antibodies from Abbkine (A25012, 1:10000, China) and CST (14708, 1:2000, USA).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

doi: 10.3389/fendo.2024.1407859

Figure Lengend Snippet: Col11a2 mRNA and protein expression in goiter mouse model. (A) Col11a2 protein expression levels increased in the 10 weeks treatment group compared with the 10 weeks control group by western blotting. (B) Col11a2 protein expression levels increased in the 20 weeks treatment group compared with the 20 weeks control group by western blotting. (C) Col11a2 expression mRNA levels increased in both treatment groups detected by qRT−PCR. (D) IHC result. (D1) Quantitative analysis of IHC results. Col11a2 expression levels increased in the 10 weeks treatment 1 group compared with 10 weeks control group. Col11a2 expression levels increased in the 20 weeks treatment group compared with the 20 weeks control group. (D2) 10 weeks control group. (D3) 20 weeks control. (D4) 10 weeks treatment. (D5) 20 weeks treatment group. Scale bar = 25 μm. * P < 0.05, ** P < 0.01. *** P < 0.001.

Article Snippet: Primary antibodies are rabbit anti-mouse Col11a2 antibody (YT1009, 1:500, Immunoway, China) and mouse anti-mouse β-actin antibody (TA811000, 1:2000, OriGene, USA) while mouse and rabbit second antibodies from Abbkine (A25012, 1:10000, China) and CST (14708, 1:2000, USA).

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Endocrinology

Article Title: A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

doi: 10.3389/fendo.2024.1407859

Figure Lengend Snippet: LNC60-Col11a2 axis in Nthy-ori-1 cells. (A) After treatment with 10 5 and 5×10 6 KIO 3 for 72 h, qPCR showed that LNC60 and Col11a2 mRNA levels were significantly increased compared with those in the control group. (B) With 5×10 6 KIO 3 treated for 72 h, Wb shows Col11a2 protein level was significant increased compared with the Control group. (C) After siLNC60, qPCR showed that LNC60 and Col11a2 were significantly decreased compared with those in the NC group. (D) After siLNC60, Wb showed that the Col11a2 protein level was significantly decreased compared with that in the NC group. (E) After siLNC60, the CCK8 assay showed a significant cell viability inhibition in the 5×10 6 M KIO 3 treatment group at 72 hours. * P <0.05, ** P <0.01, *** P <0.001, t test .

Article Snippet: Primary antibodies are rabbit anti-mouse Col11a2 antibody (YT1009, 1:500, Immunoway, China) and mouse anti-mouse β-actin antibody (TA811000, 1:2000, OriGene, USA) while mouse and rabbit second antibodies from Abbkine (A25012, 1:10000, China) and CST (14708, 1:2000, USA).

Techniques: Control, CCK-8 Assay, Inhibition

Journal: Frontiers in Endocrinology

Article Title: A new LNC89/LNC60-Col11a2 axis revealed by whole-transcriptome analysis may be associated with goiters related to excess iodine nutrition

doi: 10.3389/fendo.2024.1407859

Figure Lengend Snippet: The expression of LNC60 and Col11a2 mRNA in the peripheral blood of Goiter patients and health control populations. *** P< 0.001, **** P< 0.0001, t test.

Article Snippet: Primary antibodies are rabbit anti-mouse Col11a2 antibody (YT1009, 1:500, Immunoway, China) and mouse anti-mouse β-actin antibody (TA811000, 1:2000, OriGene, USA) while mouse and rabbit second antibodies from Abbkine (A25012, 1:10000, China) and CST (14708, 1:2000, USA).

Techniques: Expressing, Control

Journal:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Effect of overexpressing rat and human CRM1 on Rex activity. (A) REF52 cells were transfected with 0.075 μg of pSRαRex along with 0.5 μg of pDM128RxRE and 0.1 μg of pCDMβ-gal in combination with various amounts of pSRαhCRM1 (⧫) or pSRαrCRM1 (▪). The CAT/β-Gal ratios of all samples were calculated. The ratio of the sample transfected with pSRαRex in the absence of either the hCRM1 or rCRM1 expression plasmid was arbitrarily set to 1. (B) CRM1 proteins produced in REF52 cells that were transfected with 0.3 μg of either rCRM1 or hCRM1 expression plasmids (lane 1, pSRα296; lane 2, pSRαhCRM1; lane 3, pSRαrCRM1) were analyzed by Western blotting as described in Materials and Methods.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Produced, Western Blot

Journal:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Ability of hCRM1 and rCRM1 to support Rex function in HeLa cells. (A) HeLa cells were transfected with 0.05 μg of pSRαRex, 0.5 μg of pDM128RxRE, and 0.1 μg of pCDMβ-gal together with various amounts of pSRαhCRM1 or pSRαrCRM1. At 8 h posttransfection, the medium was replaced with medium containing LMB at 0.6 or 0.8 nM. At 24 h posttransfection, the cells were subjected to CAT and β-Gal measurement. The CAT/β-Gal ratios of all samples were calculated. The ratio of the control sample transfected with pSRαRex in the absence of either hCRM1 or rCRM1 expression plasmids and in the absence of LMB was arbitrarily set to 1. The amounts of CAT and the β-Gal activity in the control sample were over 300 pg and 2.5 × 10−3 U, respectively. (B) CRM1 proteins produced in HeLa cells transfected with 0.3 μg of each CRM1 expression plasmid (lane 1, pSRα296; lane 2, pSRαhCRM1; lane 3, pSRαrCRM1) were analyzed by Western blotting.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Transfection, Expressing, Activity Assay, Produced, Plasmid Preparation, Western Blot

Journal:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Interaction of Rex with either hCRM1 or rCRM1. (A) REF52 cells were transfected with the plasmid expressing either GAL-hCRM1 or GAL-rCRM1 in combination with pRexVP, pG5BLuc, and pCDMβ-gal. The Luc/β-Gal ratios of all samples were calculated. GAL-, plasmid expressing only the GAL4 region, which was used as a negative control. (B) Western blot of GAL-hCRM1 and GAL-rCRM1 proteins synthesized in transfected REF52 cells. (C) Pull-down assay using recombinant Rex protein. Purified His-Rex(g10) proteins immobilized on chelating Sepharose were incubated with HeLa or REF52 cell extract in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of GTP-charged recombinant RanQ69L protein. The sample of lane 1 contained neither cell lysate nor recombinant RanQ69L proteins during incubation. The volume of the cell extract subjected to the binding reaction was nine times of that of the input fraction.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Transfection, Plasmid Preparation, Expressing, Negative Control, Western Blot, Synthesized, Pull Down Assay, Recombinant, Purification, Incubation, Binding Assay

Journal:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Rex-Rex dimerization analyzed by the coimmunoprecipitation method. HeLa and REF52 cells were transfected with the plasmids which express GAL-Rex and Rex-VP or their derivatives as indicated. At 48 h posttransfection, the cells were harvested and the supernatants were incubated with anti-VP antibodies immobilized on protein-G Sepharose in the presence of GTP-charged recombinant RanQ69L. The recombinant hCRM1 protein was added to the sample, which was derived from the REF52 cells transfected with GAL-Rex and Rex-VP (lane 6) during incubation. The volume of the cell extract subjected to the coimmunoprecipitation reaction was nine times of that of input fraction. Rabbit anti-Rex C terminus antibodies were used for Western blotting.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Transfection, Incubation, Recombinant, Derivative Assay, Western Blot