Journal: Angiogenesis

Article Title: αB-crystallin/HspB5 regulates endothelial–leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin

doi: 10.1007/s10456-013-9367-4

Figure Lengend Snippet: Ectopic expression of αB-crystallin increases E-selectin levels in HUVEC. a Representative Western Blot for αB-crystallin in HUVEC transduced with a lentiviral vector coding for full-length human αB-crystallin ( pgk:cryab ) or an empty control vector ( pgk:ev ). Actin served as loading control. b Representative FACS plots of E-selectin staining on HUVEC transduced with pgk:cryab and pgk:ev after treatment with TNF-α for 24 h. c Quantification of surface expression of E-selectin in HUVEC transduced with pgk:cryab ( white bars ) or empty vector controls ( black bars ). Cells were treated with TNF-α for 5 h ( left panel ) or 24 h ( right panel ) d HUVEC transduced with pgk:cryab ( white bars ) or empty vector controls ( black bars ) were stimulated for 3, 6, 12 and 24 h with TNF-α and expression of E-selectin relative to hprt was determined by qPCR ( Bars represent mean ± SD fold expression compared to pgk:ev after 3 h of TNF-α (normalized data from 3 independent experiments), * = p < 0.05). e HUVEC transduced with pgk:cryab ( white bars ) or empty vector controls ( black bars ) were grown to confluency and stimulated with TNF-α for 24 h before co-incubation with Jurkat cells for 15 min. Firmly adherent cells were microscopically quantified. ( Bars represent mean ± SD (normalized data from 3 individual independent), * = p < 0.05)

Article Snippet: For synthesis of the lentivirus vector pgk:cryab , a full length clone of human cryab cDNA (NM_001885.1, Origene, Rockville, MD) was cloned into a modified lentiviral pgk vector [ ] containing an internal ribosomal entry site (IRES) and eGFP as selection marker.

Techniques: Expressing, Western Blot, Transduction, Plasmid Preparation, Control, Staining, Incubation

Journal: Angiogenesis

Article Title: αB-crystallin/HspB5 regulates endothelial–leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin

doi: 10.1007/s10456-013-9367-4

Figure Lengend Snippet: TNF-α-induced endothelial activation is reduced in endothelial cells derived from cryab -/- mice. a Representative western blot showing αB-crystallin expression in MyEnd wild type and MyEnd cryab - / - cells. b , c , d MyEnd wild type and MyEnd cryab - / - cells were treated with TNF-α for 3, 6, 18 and 24 h and expression of E-selectin ( b ), ICAM-1 ( c ) and VCAM-1 ( d ) relative to hprt was determined by qPCR. ( Bars represent mean ± SD (normalized data from 3 independent experiments), * = p < 0.05)

Article Snippet: For synthesis of the lentivirus vector pgk:cryab , a full length clone of human cryab cDNA (NM_001885.1, Origene, Rockville, MD) was cloned into a modified lentiviral pgk vector [ ] containing an internal ribosomal entry site (IRES) and eGFP as selection marker.

Techniques: Activation Assay, Derivative Assay, Western Blot, Expressing

Journal: Angiogenesis

Article Title: αB-crystallin/HspB5 regulates endothelial–leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin

doi: 10.1007/s10456-013-9367-4

Figure Lengend Snippet: TNF-α-induced activation of NF-κB is reduced in the absence of αB-crystallin. MyEnd wt and MyEnd cryab - / - cells were treated with TNF-α for 15, 30, 45 min and 1 h, protein lysates were prepared and expression of IκB was determined by western blot. One representative blot of three independent experiments is shown

Article Snippet: For synthesis of the lentivirus vector pgk:cryab , a full length clone of human cryab cDNA (NM_001885.1, Origene, Rockville, MD) was cloned into a modified lentiviral pgk vector [ ] containing an internal ribosomal entry site (IRES) and eGFP as selection marker.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Angiogenesis

Article Title: αB-crystallin/HspB5 regulates endothelial–leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin

doi: 10.1007/s10456-013-9367-4

Figure Lengend Snippet: In vivo leukocyte–endothelial interactions are altered in cryab - / - mice, associated with decreased expression of endothelial adhesion molecules. Intravital microscopy was used to analyze leukocyte–endothelial interactions in inflamed venules of the cremaster muscle of wild type ( white bars ) and cryab - / - mice ( black bars ). Analysis of rolling velocity ( a ) and rolling flux ( b ) 3.5, 4, and 4.5 h after intrascrotal injection of TNF-α (n = 5, mean ± SD, * p < 0.05). qPCR analysis of mRNA expression of ICAM-1 ( c ), VCAM-1 ( d ) and E-selectin ( e ) relative to hprt in mouse cremaster muscles harvested 4.5 h after intrascrotal injection of TNF-α (n = 4, mean ± SD, * p < 0.05)

Article Snippet: For synthesis of the lentivirus vector pgk:cryab , a full length clone of human cryab cDNA (NM_001885.1, Origene, Rockville, MD) was cloned into a modified lentiviral pgk vector [ ] containing an internal ribosomal entry site (IRES) and eGFP as selection marker.

Techniques: In Vivo, Expressing, Intravital Microscopy, Injection, Muscles

Journal: Scientific reports

Article Title: Gestational high-sucrose diet mediated vascular hyper-contractility in mesenteric arteries from offspring.

doi: 10.1038/s41598-025-93361-2

Figure Lengend Snippet: Fig. 4. The RNA-Seq analysis of MAs from offspring. A Heat map of RNA-Seq. CON: n = 3, HS: n = 3. B Volcano map of RNA-Seq. CON: n = 3, HS: n = 3. C The mRNA expressions of APCDD1, HSPE1 and CRYAB. D The protein expressions of APCDD1, HSPE1 and CRYAB. E KEGG pathway enrichment analyses. n = 3–7 from different litters. *p < 0.05, **p < 0.001, ****p < 0.0001.

Article Snippet: Following blocking non-specific binding sites by 5% skim milk, the PVDF membranes were incubated with the primary antibodies, including GAPDH (1:2000, Affinity, China), β-actin (1:5000, Affinity, China), Wnt5a (1:1000, Affinity, China), PKCα (1:1000, Abcam, US), CRYAB (1:1000, Absin, China), HSPE1 (1:1000, Absin, China), and APCDD1 (1:1000, Proteintech, China).

Techniques: RNA Sequencing

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: A The quantitative results of the Pearson’s Correlation Coefficient (PCC). B The Violin plot showing the distribution and differences in the intensity values of modification sites among different samples. C The expression heatmap of differentially modified sites, where red represents high expression, blue represents low expression, and gray represents non-quantifiable values in the corresponding sample. D–G The GO and KEGG pathway enrichment analyses of lactylation-activated proteins are presented via bubble plots (where D: MF, molecular function; E: BP, biological process; F: CC, cellular component; G: pathway enrichment). H Functional annotation of identified proteins. I The Volcano plot of differentially modified proteins and sites between LF/HLF groups. The information of the top five differentially modified sites is also marked in the figure. J The MS/MS spectra including C-terminal y-ions and N-terminal b-ions of CRYAB_K92.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques: Modification, Expressing, Functional Assay, Tandem Mass Spectroscopy

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: A The co-IP showed the lactylation level of CRYAB in LF/HLF tissue. B The co-IP showed the regulation of exogenous lactate on the lactylation level of CRYAB in HLF cells. C The co-IP showed the lactylation level of CRYAB in HLF cells before and after the mutation at the CRYAB _K92 site. D , E The western blot and the quantification analysis showed the regulatory effect of exogenous lactate combined with the mutation at the CRYAB _ Kla92 on the fibrosis level in HLF cells. F , G The western blot and the quantification analysis showed the efficiency of P300 silencing and overexpression. H The co-IP showed the regulation of cellular lactylation level of CRYAB by the site mutation in combination with the lactylation-modifying enzyme P300 and the P300 inhibitor C646. I , J The western blot and the quantification analysis showed the regulatory effects on the fibrosis level of HLF cells before and after CRYAB _K92 mutation, as well as in combination with P300 and C646. K–M The results of EDU and FCM assays showing the regulation of the proliferation and cell cycle of HLF cells by the mutation at the CRYAB _K92 site in combination with P300 and C646. Scale bar=50 μm. O The co-IP results compared the regulatory effects of K92 site mutation, P300 knockout, and the use of the P300 inhibitor C646 on CRYAB lactylation in HLF cells. P , Q The western blot and the quantification analysis results compared the regulatory effects of K92 site mutation, P300 knockout, and the use of the P300 inhibitor C646 on the fibrosis level of HLF cells. The quantitative data are presented as mean ± SD, with individual data points overlaid to show the distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Over Expression, Knock-Out

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: A The scatter plot shows differentially expressed proteins following mutation of the Kla92 site. S100A16 expression was significantly downregulated after the mutation. B Molecular docking prediction between S100A16 and CRYAB. The binding pose with the lowest PIPER pose energyis displayed. C The sensorgram of Surface plasmon resonance (SPR) analysis confirms a specific and direct interaction between S100A16 and CRYAB. D , E The western blot and quantitative analysis show that the protein half-life of S100A16 was significantly shortened after the CRYAB_K92 mutation. F The qRT-PCR results showed the mutation of the CRYAB _K92 site significantly reduced the mRNA expression level of S100A16 in HLF cells. G Actinomycin D chase analysis of S100A16 mRNA stability in CRYAB _K92 WT and Mut cells. mRNA levels at each time point were normalized to the respective 0-hour value (set as 100%). H , I The cellular immunofluorescence showed that after the CRYAB _K92 mutation, the signal levels of both lactylation (Kla, red) and S100A16 (green) decreased in HLF cells. The merged image shows co-localization between Kla and S100A16 signals. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. J , K The cellular immunofluorescence showed that the mutation of CRYAB _K92 led to reduced expression levels of both CRYAB (red) and S100A16 (green) in HLF cells. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. L The tissue immunofluorescence analysis showed that compared with LF tissue, S100A16 (red) expression was significantly increased in HLF tissue. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. M The co-IP assays using a CRYAB antibody confirmed that the CRYAB _K92 mutation significantly reduced the level of S100A16 protein that interacts with CRYAB. The quantitative data are presented as mean ± SD, with individual data points overlaid to show the distribution. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques: Mutagenesis, Expressing, Binding Assay, SPR Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: A , B The results of western blot analysis showed the regulation of CRYAB_K92 in cooperation with S100A16 on the fibrosis and the RAGE pathway. C–F The results of EDU and FCM indicate that CRYAB_K92 in cooperation with S100A16 can promote the proliferation of HLF cells. Scale bar = 50 μm. G , H The results of western blot showed the regulation of CRYAB _K92 and S100A16 on the fibrosis of HLF cells and the RAGE pathway in combination with NaLA. I The lactic acid content in HLF cells treated with CRYAB _K92 site mutation, exogenous lactic acid, and S100A16 silencing. J The ATP production in HLF cells treated with CRYAB _K92 site mutation, exogenous lactic acid, and S100A16 silencing. K The ECAR in HLF cells treated with CRYAB _K92 site mutation, exogenous lactic acid, and S100A16 silencing. The quantitative data are presented as mean ± SD, with individual data points overlaid to show the distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques: Western Blot, Mutagenesis

Journal: Communications Biology

Article Title: CRYAB_K92 lactylation drives hypertrophy of the ligamentum flavum via an S100A16/RAGE-mediated glycolysis-fibrosis positive feedback loop

doi: 10.1038/s42003-026-10103-4

Figure Lengend Snippet: The hyperlactate state and lactylation of CRYAB_K92 promote fibrosis and hyperproliferation of ligamentum flavum cells by upregulating S100A16 and activating the RAGE pathway.

Article Snippet: Recombinant S100A16 (MedChemExpress) was immobilized on a CM5 sensor chip, and serially diluted recombinant CRYAB (MedChemExpress) was injected as the analyte.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: a study of DNAJB6 chaperone

doi: 10.3389/fnmol.2015.00040

Figure Lengend Snippet: Canonical members of HSPB family prevent the aggregation of Aβ-GFP. Cells were transfected with HSPB1, HSPB5, HSPB7 or FRTTO at 1:3 ratio for 48 h. Quantification of the pellet/soluble ratio of Aβ-GFP relative to FRTTO was depicted in the chart above the blot. Values represent mean ± SE of two independent experiments. ** P < 0.01, ns = non significant.

Article Snippet: The membranes were blocked with 5% dry milk in PBS with 0.1% Tween 20 (PBST) for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: 6E10 (1:1000 in TBST, Covance), anti V5 (1:5000 in PBST, Invitrogen), anti β-actin (1:1000 in PBST, Abcam), anti HSPB1 (1:1000 in PBST, Stress Marq Biosciences), anti HSPB5 (1:1000 in PBST, Stress Marq Biosciences) and anti HSPB7 (1:1000, Abnova).

Techniques: Transfection

Journal: Oncotarget

Article Title: Phosphorylation and degradation of αB-crystallin during enterovirus infection facilitates viral replication and induces viral pathogenesis

doi: 10.18632/oncotarget.20366

Figure Lengend Snippet: HL-1 mouse cardiomyocytes (A-D) and rat neonatal cardiomyocytes (E) were transduced with adenoviral HA-CryAB-WT or control vector (GFP) for 48 hours, and then infected with CVB3 for 24 hours. (A) Representative images of sham and CVB3-infected HL-1 cells overexpressing HA-CryAB-WT or GFP control as indicated. (B) Cell viability was determined by the MTS assay and presented as percentage changes as compared with sham infection that was arbitrarily set as 100% (mean ± SD, n=3). (C, E) Western blotting was carried out to examine protein levels of viral capsid protein VP1, HA-tagged CryAB, and β-actin/GAPDH (loading control) in HL-1 (C) and rat neonatal cardiomyocytes (E). VP1 levels were quantitated by densitometric analysis, normalized to β-actin, and presented underneath as fold changes compared with the GFP-transduced, CVB3-infected cells. (D) Plaque assay was performed to measure CVB3 viral progeny titer in supernatants collected from CVB3-infected HL-1 cells transduced with HA-CryAB-WT or GFP control vector (mean ± SD, n=3).

Article Snippet: The primary antibodies used in this study include antibodies against VP1 (DakoCytomation, #M706401-1), desmin (Cell Signaling, #5332), CryAB (StressMarq, #SMC-165), p-Ser19-CryAB (Enzo Life Sciences, #ADI-SPA-225), p-Ser45-CryAB (Enzo Life Sciences, #ADI-SPA-226), p-Ser59-CryAB (Enzo Life Sciences, #ADI-SPA-227), HA (Roche, #11867423001), sequestosome 1/p62 (PROGEN Biotechnik GmbH, #GSQSTM1-C), and p53 (Santa Cruz Biotechnology, #sc-126).

Techniques: Transduction, Plasmid Preparation, Infection, MTS Assay, Western Blot, Plaque Assay