Journal: Comparative Hepatology

Article Title: CIDE-A is expressed in liver of old mice and in type 2 diabetic mouse liver exhibiting steatosis

doi: 10.1186/1476-5926-6-4

Figure Lengend Snippet: CIDE-A Northern and Immunoblot analyses . A) Northern analysis of RNA extracted from normal (C) and type 2 diabetic (D) liver and heart tissue. Total RNA (10 μg) from the appropriate tissues was resolved by denaturing agarose gel electrophoresis, transferred to positively charged nylon membrane, hybridized with the [α- 32 P]dCTP-labeled mouse CIDE-A cDNA and exposed to Bio-Max MR film. Ethidium bromide stain of RNA (10 μg/lane) prior to transfer to nylon membrane. The values represent the level of CIDE-A gene expression for the individual tissue as determined by DNA microarray analysis (ND: not determined). The approximated size (1.3 kb) of the CIDE-A mRNA is noted on the right. B) Immunoblot demonstrating increased CIDE-A protein levels in type 2 diabetic mouse liver. Sixty μg of liver and heart extract was electrophoresed on a 12.5% SDS-polyacrylamide gel and the resolved proteins transferred to a nitrocellulose membrane. The membrane was immunoblotted using a rabbit anti-mouse CIDE-A polyclonal antibody and a goat anti-rabbit IgG polyclonal antibody conjugated to horseradish peroxidase. Arrow indicates mouse CIDE-A. The values represent the level of CIDE-A gene expression for the individual tissue as determined by DNA microarray analysis (ND: not determined).

Article Snippet: Resolved proteins were transferred to a nitrocellulose membrane and immunoblotted using a rabbit anti-mouse CIDE-A polyclonal antibody (QED Bioscience Inc., San Diego, CA) as previously described [ ].

Techniques: Northern Blot, Western Blot, Agarose Gel Electrophoresis, Membrane, Labeling, Staining, Gene Expression, Microarray

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: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: List of Antibodies Used in This Study

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques:

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Screening of Hsps using double-label FIHC with different anti-Hsp and anti-α-syn antibodies visualized by Alexa Fluor 594 or Texas Red and Alexa Fluor 488, respectively, shows co-localization of a subset of Hsps examined in LBs (a–g) and GCIs (h–n). Yellow color represents co-localization of Hsp and α-syn. Hsp27 (b), Hsp40 (c), Hsc70 (e), and Hsp90 (g) are located in LBs, whereas αB-crystallin (h) and Hsp90 (n) are located in GCIs and threads. Specificity of Hsp90 antibodies (o), 9D2 (lanes 1 and 2), and AC88 (lanes 3 and 4) is shown in HS fraction of human (lanes 1 and 3) and mouse (lanes 2 and 4) brain homogenates. Rat 9D2 does not recognize mouse Hsp90 (lane 2). Photomicrographs in p–x show modest Hsp90 IR in neurons of normal brain (p), which also is seen in α-synucleinopathy brains, in addition to more intense Hsp90 IR in LBs (arrowhead) of the PD SN (q), DLB amygdala (r), and GCIs (arrow) in the MSA pons (s). Little or no iHsp90 IR is seen in neurofibrillary tangles of AD (t, double arrowheads) and DLB (r). t: IHC profile with an antibody against Hsp90 (9D2) or tau (AT8) (inset) from adjacent sections in the CA1 region of AD hippocampus are presented for comparison. It is apparent that Hsp90 IR is reduced in neurofibrillary tangles. u: Modest Hsp90 IR is noted in ubiquitin inclusions in the hippocampus of the FTD-MND brain. Inset shows ubiquitin IR in inclusions on the adjacent section. v and w show double-label IHC with horseradish peroxidase-DAB and β-galactosidase-X-gal to illustrate co-localization of iHsp90 IR (brown) with α-syn IR (blue) in LBs (arrowhead) in the midbrain of PD (v) as well as GCIs (arrow) in the pons of MSA (w). x: No CHIP IR was found on GCI in the pons of MSA, whereas the adjacent section indicates descent α-syn IR inclusions (inset). Scale bars, 10 μm.

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques:

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Double-label FIHC confirms Hsp90 IR (Texas Red) co-localizes with α-syn IR (Alexa Fluor 488) in LBs of PD amygdala (arrowhead in a–c) and GCIs in the MSA pons (d–f). g–j: Representative images from triple-label FIHC of DLB amygdala shows differential co-localization of Hsp90 with α-syn (arrow) rather than tau-positive inclusions (arrowhead) in these preparations. Even when tau IR is located in iHsp90 IR cells, the subcellular localization of these proteins is normally discordant (asterisk). k: Semiquantitative analysis reveals the number of iHsp90 IR with α-syn- or tau IR lesions including intracytoplasmic inclusions, dystrophic neurites, and spheroids. l: iHsp90 co-localizes with α-syn to a significantly greater extent (P < 0.01) than tau in disease lesions. Asterisks indicate statistically significant difference. P < 0.05. Scale bars, 10 μm (a–j).

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques:

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Triple-label FIHC micrographs of iHsp90 (Texas Red), ubiquitin (Alexa Fluor 488), and α-syn (AMCA) of DLB amygdala (a–d) and MSA pons (e–h) indicate that iHsp90 co-localizes preferentially with ubiquitinated α-syn lesions (arrow), whereas a subset of α-syn-positive profiles are ubiquitin-negative (arrowhead). i and j: Data from semiquantitative analyses of triple-label FIHC studies of amygdala from DLB brain (n = 6 in i) and pons of MSA brains (n = 6 in j) showing that a similar percentage (∼30 to 45%) of the total number α-syn lesions (neurites plus perikaryal inclusions) are iHsp90− (α-syn + Hsp90) and ubiquitin IR (α-syn + Ubiq), whereas >90% of perikaryal α-syn inclusion lesions are iHsp90− (α-syn + Hsp90) and ubiquitin IR (α-syn + Ubiq). There were no significant differences between the number of Hsp90-positive and ubiquitin-positive α-syn inclusions. Scale bars, 10 μm (a–h).

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques:

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Representative images from Western blot analyses (WB) of HS, RIPA, and FA samples of cingulate cortex from normal (norm), PD (PD), DLB brains (DLB), and pons of MSA brain (MSA). Equal amounts of sample were analyzed by SDS-polyacrylamide gel electrophoresis. These results show that the FA fraction from PD, DLB, and MSA brains harbors accumulations of oligomeric α-syn species, while variably increased amounts of Hsp90 and Hsc70, but not Hsp70, are seen in FA fractions of all α-synucleinopathy disease brains. Additionally, some pathological accumulations of Hsp40 as well as Hsp27 and αB-crystallin were noted in PD/DLB and MSA brains. Polyubiquitinated protein species in the FA fraction were more apparent in the disease brains than normal brain.

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: A: Effects of MG-132 and lactacystin (LC) on cultured cells. In oligodendrocyte primary culture (a and b), exposure to MG-132 (1 μmol/L) for 3, 5, 7, and 18 hours leads to the appearance of polyubiquitinated proteins after 18 hours, with slight up-regulation of Hsp90 levels (a). By 18 hours of incubation of different doses of MG-132 (1, 5, 10 μmol/L) or LC at one (10 μmol/L) dose there is a sharp induction of ubiquitinated proteins, accompanied by up-regulation of Hsp90 protein and mRNA level (b, bottom). Similar results are seen in OLN (c) cells transfected with tau40 plus α-syn and N2A cells (d) transfected with α-syn although the levels of tubulin and α-syn remain unchanged. Bar graphs indicate quantitation of variable Hsp90 protein levels due to MG132 treatment in respective cell types, confirming up-regulation of the protein level in all cases. B: Co-immunoprecipitation analysis using OLN-tau40-α-syn cells revealed noticeable protein-protein interactions between α-syn and Hsp90 as well as Hsc70 (bound), but not between α-syn and Hsp70 or Hsp40 (unbound). The protein complex of interest was isolated from the OLN cell homogenate using either no antibody (no Ab), α-syn Ab, Hsp90, Hsc70, Hsp70, or Hsp40 (IP Ab) and detected by SNL1 (α-syn) or AC88 (Hsp90) by Western blot (WB Ab). Input represents original material.

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques: Cell Culture, Incubation, Transfection, Quantitation Assay, Immunoprecipitation, Isolation, Western Blot

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: In the brainstem of a 3-month-old control non-Tg mouse (a–c), M7 Tg mouse (data not shown), and asymptomatic α-syn (M83) Tg mouse (d–f), there are no α-syn lesions and modest Hsp90 IR. However, in older symptomatic M83 Tg mouse (9 months old), numerous α-syn inclusions are formed, which are frequently associated with iHsp90 IR (g–i, arrow) and ubiquitin IR (j–l, arrow). Western blots of non-Tg (lanes 1 to 3 in m–o) and M83Tg mouse (lanes 4 to 6 in m–o) show accumulation of α-syn (m), Hsp90, and Hsc70, but not Hsp70, Hsp40, or αB-crystallin (n) and polyubiquitinated (o) proteins (likely including α-syn) exclusively in the FA fraction (lane 6) of 9-month-old symptomatic M83 Tg mouse brain, but not in the 3-month-old non-Tg mouse brain (m–o) or 9-month-old non-Tg mouse (data not shown). Scale bars, 10 μm (a–l).

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques: Western Blot

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: Double-immunoelectron microscopy reveals a close association of Hsp90 and α-syn in filamentous LBs in dopaminergic neurons of the SN in PD brain (a, c, e). α-Syn-positive (10 nm gold, arrow) filaments in the LB are also Hsp90-positive (18 nm gold, arrowhead), whereas unaffected dopaminergic neurons in the same SN section (f) reveal infrequent Hsp90 labeling (arrowhead). Image e is a high-power view of the inset in c, from the center of a LB. PG, neuronal pigment; N, nucleus.

Article Snippet: 45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, α-syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.

Techniques: Immuno-Electron Microscopy, Labeling