Journal: Cell Death & Disease

Article Title: TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity

doi: 10.1038/s41419-020-2626-6

Figure Lengend Snippet: a BAG-1 expression after miR-342 overexpression/inhibition was addressed by western blot. Results were normalized with α-tubulin and compared with the respective controls (mean ± SD, n = 6). To evaluate the involvement of BAG-1 on NF-kB activation, N9 microglia were transfected with a siRNA to silence b or with a plasmid (1ug/mL) to overexpress BAG-1 c . BAG-1 and ph-NF-kB p65 expression levels were evaluated by western blot. Results were normalized with GAPDH and compared with the respective controls (mean ± SD, n = 2–4). Statistical significance: * p < 0.05, Wilcoxon matched-pairs test.

Article Snippet: Overexpression of BAG-1 in N9 microglia was achieved by transiently transfecting the cells with a BAG-1 mammalian expression vector (pCMV6-BAG-1, Origene).

Techniques: Expressing, Over Expression, Inhibition, Western Blot, Activation Assay, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity

doi: 10.1038/s41419-020-2626-6

Figure Lengend Snippet: We found miR-342 to be upregulated in microglia activated with TNF-α. miR-342 promotes NF-kB activation by inhibiting BAG-1, leading to the overexpression of pro-inflammatory mediators, including TNF-α, in a positive feedback loop, possibly perpetuating microglia activation. Importantly, inhibition of miR-342 attenuated TNF-α-driven microglia activation. Moreover, microglia activation by miR-342 led to increased neurotoxicity with high levels of nitrites being detected in co-cultures supernatants.

Article Snippet: Overexpression of BAG-1 in N9 microglia was achieved by transiently transfecting the cells with a BAG-1 mammalian expression vector (pCMV6-BAG-1, Origene).

Techniques: Activation Assay, Over Expression, Inhibition

Journal: Genes

Article Title: BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation

doi: 10.3390/genes13081338

Figure Lengend Snippet: Primer sequences used in the present study.

Article Snippet: To detect the interactions between HSP70 and HSPB8 and BAG3 protein, the membranes were incubated with antibodies against HSP70 (66183-1-Ig, ProteinTech Group, Inc., Chicago, IL, USA) and HSPB8 (15287-1-AP, ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: Sequencing

Journal: Genes

Article Title: BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation

doi: 10.3390/genes13081338

Figure Lengend Snippet: Overexpression of BAG3 reduces atherosclerotic lesions in ApoE −/− mice. ( A – D ) The levels of serum TG, TC, HDL-C and LDL-C in mice ( n = 6). ( E ) Hematoxylin–eosin (H&E) and Oil Red O staining of aortic root sections showing the atherosclerotic lesions and lipid deposition (scale bar indicates 200 μm) ( n = 3). ( F , G ) Representative en face images of Oil Red O staining of aortas; the lesion area of the whole aorta was quantified ( n = 3). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Article Snippet: To detect the interactions between HSP70 and HSPB8 and BAG3 protein, the membranes were incubated with antibodies against HSP70 (66183-1-Ig, ProteinTech Group, Inc., Chicago, IL, USA) and HSPB8 (15287-1-AP, ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: Over Expression, Staining

Journal: Genes

Article Title: BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation

doi: 10.3390/genes13081338

Figure Lengend Snippet: BAG3 prevents EndMT in vivo and vitro. ( A ) Morphological changes post the indicated treatments were observed under the microscope (scale bar indicates 100 µm) ( n = 3). ( B – E ) CD31, BAG3 and α-SMA protein expressions were detected using Western blotting. Fold changes are shown ( n = 5). ( F , G ) Transwell cell invasion assay was performed to analyze the migration ability of cells (scale bar indicates 100 µm) ( n = 3). ( H ) Representative images of double-fluorescent staining with α-SMA (green) and CD31 (red) in the endothelium. The nuclei were stained blue with DAPI. (scale bar indicates 25 μm) ( n = 3). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Article Snippet: To detect the interactions between HSP70 and HSPB8 and BAG3 protein, the membranes were incubated with antibodies against HSP70 (66183-1-Ig, ProteinTech Group, Inc., Chicago, IL, USA) and HSPB8 (15287-1-AP, ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: In Vivo, Microscopy, Western Blot, Invasion Assay, Migration, Staining

Journal: Genes

Article Title: BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation

doi: 10.3390/genes13081338

Figure Lengend Snippet: BAG3 positively regulates autophagy in HUVECs. ( A – C ) The LC3-II/LC3-I ratio and p62 protein expressions were detected using Western blotting. Fold changes are shown ( n = 5). ( D ) Electron micrographs of HUVECs submitted to the indicated treatments, including autophagosomes and autolysosomes (red arrow) (scale bar indicates 2 µm and 500 nm) ( n = 3). ( E ) Representative images and quantification of GFP-mRFP-LC3II puncta in HUVECs. (scale bar indicates 25 µm) ( n = 5). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Article Snippet: To detect the interactions between HSP70 and HSPB8 and BAG3 protein, the membranes were incubated with antibodies against HSP70 (66183-1-Ig, ProteinTech Group, Inc., Chicago, IL, USA) and HSPB8 (15287-1-AP, ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: Western Blot

Journal: Genes

Article Title: BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation

doi: 10.3390/genes13081338

Figure Lengend Snippet: BAG3 suppresses EndMT by inducing autophagy in HUVECs. ( A – D ) CD31 and α-SMA protein expressions and the LC3-II/LC3-I ratio were detected using Western blotting. Fold changes are shown ( n = 5). ( E , F ) Transwell cell invasion assay was performed to analyze the migration ability of cells (scale bar indicates 100 µm) ( n = 3). ( G ) Representative images of double-fluorescent staining with α-SMA (green) and CD31 (red). The nuclei were stained blue with DAPI. (scale bar indicates 50 µm) ( n = 3). The data are presented as the mean ± S.E.M. * p < 0.05, ** p < 0.01.

Article Snippet: To detect the interactions between HSP70 and HSPB8 and BAG3 protein, the membranes were incubated with antibodies against HSP70 (66183-1-Ig, ProteinTech Group, Inc., Chicago, IL, USA) and HSPB8 (15287-1-AP, ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: Western Blot, Invasion Assay, Migration, Staining

Journal: Genes

Article Title: BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation

doi: 10.3390/genes13081338

Figure Lengend Snippet: BAG3 blocks ox-LDL-induced EndMT through formation of CASA complex with HSP70 and HSPB8 in HUVECs. ( A ) Co-IP analysis for the interaction of BAG3 with HSP70 or HSPB8. HUVEC extracts were immunoprecipitated with anti-BAG3 antibody and probed with anti-HSP70 or anti-HSPB8 antibody. ( n = 3). ( B – H ) BAG3, HSP70, HSPB8, CD31, and α-SMA protein expressions and the LC3-II/LC3-I ratio were analyzed using Western blotting. Fold changes are shown ( n = 5). ( I ) Morphological changes post the indicated treatments were observed under a microscope (scale bar indicates 100 µm). ( J , K ) Transwell cell invasion assay was performed to analyze the migration ability of cells (scale bar indicates 100 µm). ( n = 3). ( L ) Representative images of double-fluorescent staining with α-SMA (green) and CD31 (red). The nuclei were stained blue with DAPI (scale bar indicates 50 µm). The data are presented as the mean ± S.E.M. ns p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: To detect the interactions between HSP70 and HSPB8 and BAG3 protein, the membranes were incubated with antibodies against HSP70 (66183-1-Ig, ProteinTech Group, Inc., Chicago, IL, USA) and HSPB8 (15287-1-AP, ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Microscopy, Invasion Assay, Migration, Staining

Journal: Cell Death Discovery

Article Title: Neuromodulation of BAG co-chaperones by HIV-1 viral proteins and H 2 O 2 : implications for HIV-associated neurological disorders

doi: 10.1038/s41420-021-00424-0

Figure Lengend Snippet: A Immunoblot results indicated H 2 O 2 treatment caused time and dosage-dependent decreases in neuronal BAG3. B qPCR demonstrated H 2 O 2 treatment causes significant decreases in neuronal BAG3 RNA. C In addition to validating H 2 O 2 -induced reductions in BAG3 protein, immunocytochemistry suggested neurons exposed to H 2 O 2 treatment may develop minor dystrophies in their branching processes. D Immunoblotting revealed H 2 O 2 reduced other BAG family members (BAG2, BAG5, BAG6), and exacerbated the reduction in BAG3 observed in response to Tat or Nef. *** p < 0.001. Scale bar equals 50 μM; ×40 magnification. Scale bar equals 20 μM; ×63 magnification.

Article Snippet: The following primary antibodies were used: ANT (Santa Cruz; SC11433), β-Tubulin (Sigma; T8578), Bcl-2 (Sant Cruz; SC7382), BAG1 (Santa Cruz; SC939), BAG2 (Novus Biologicals; NBP159086), BAG3 (Proteintech; 105991AP), BAG5 (Proteintech; 266281AP), BAG 6 (R&D Systems; AF6438), BAX (Santa Cruz; SC493), GAPDH (Santa Cruz; SC32233), Hsc/Hsp 70 (Sant Cruz; SC24), LC3 (Santa Curz; L8918), Nef (Abcam; ab42355), Tat (NIH Aids Reagent Program; R705), VDAC1 (Santa Cruz; SC8017).

Techniques: Western Blot, Immunocytochemistry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteostasis network response to environmental chronic stress: linking survival to protein aggregation in a human neuroblastoma cellular model

doi: 10.1007/s00018-025-05884-6

Figure Lengend Snippet: Analysis of the BAG1/BAG3 triage system. a Western blot analysis of the expression of BAG1 in the RIPA-soluble protein fraction after 24 h ( n = 3) and b 72 h ( n = 4). c Analysis of the expression of soluble BAG3 after 24 h ( n = 4) and d 72 h ( n = 4). e. Analysis of BAG3 in the insoluble fraction after 72 h of treatment ( n = 3). Data were normalized against Vinculin as loading control. For the insoluble fraction, the same amount of proteins quantified in the respective soluble fractions were considered. Data are expressed as fold increase ± S.E.M and compared to normalized control, shown as dashed line. Statistical significance was determined after one-way ANOVA with Dunnet’s multiple comparison test of each treatment against control. *** p < 0.001; ** p < 0.01; * p < 0.05

Article Snippet: Primary antibodies utilized were: PARP-1, Cell Signaling Technology, 9542; BiP/Grp78, Abcam, ab32618; p-eIF2⍺, Cell Signaling Technology, 9721; eIF2⍺, Cell Signaling Technology, 9722; Ubiquitin, antibodies.com, A85455; BAG3, Invitrogen, MA5-32706; BAG1, Proteintech, 19064–1-AP; LC3, RBC Lifescience, PD014; p62/SQSTM1, Cell Signaling Technology, 8025; pTDP-43, Proteintech, 80007–1-RR −1-AP; TDP-43, Proteintech, 12892–1-AP; β-Actin, Sigma-Aldrich, A5316; Vinculin, Sigma-Aldrich, V9131.

Techniques: Western Blot, Expressing, Control, Comparison