Journal: Nature communications

Article Title: A biosensor-based framework to measure latent proteostasis capacity.

doi: 10.1038/s41467-017-02562-5

Figure Lengend Snippet: Fig. 6 Probing changes in proteostasis through selective activation of different chaperone systems. a Investigation of the effect of induction of the heat shock response by HSF1 overexpression or selective overexpression of HSP90. Shown on left are western blots of the HEK293T cells matched for total protein via a BCA assay. These cells were co-transfected with HSF1 (or myc-tagged HSP90 or control of (non-fluorescent) Y66L Emerald fluorescent protein) and the L89G barnase biosensor (which also contains a myc tag). The right graphs show the Lower-slope and A50% analyses of these treatments and one or three of the biosensor variants as indicated vs. the wild-type* variant. Bars indicate means ± SEM. The right panels were analysed via a two-way ANOVA subjected to a Dunnett’s post-hoc test, results coded as ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = > 0.05. b Same paradigm as panel a. In this case, BAG1 was cotransfected with the barnase biosensor, HSP40 (DNAJB1) and HSP70 (HSPA1A). The right panels show the proportional dosage of each construct (by mass of DNA) in the transfection. Bars indicate means ± SEM. The right panel Lower-slope graph was analysed via a two-way ANOVA subjected to Dunnett’s post hoc test and the A50% graph was analysed via a one-way ANOVA subjected to a Tukey’s post-hoc test. Results coded as ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = > 0.05

Article Snippet: Plasmids expressing HSPA1A, DNAJB1 and mCherry were prepared as described previously35,36. pCMV6-AC BAG1 (cat# SC319483), pCMV6-AC HSF1 (cat# SC321225) and pCMV6-Entry myc-HSP70AA1 (cat# RC212496) were purchased from Origene.

Techniques: Activation Assay, Over Expression, Western Blot, BIA-KA, Transfection, Control, Variant Assay, Construct

Journal: Cancer gene therapy

Article Title: Functionality of hypoxia-induced BAX expression in a human glioblastoma xenograft model.

doi: 10.1038/sj.cgt.7700814

Figure Lengend Snippet: Figure 2 HIF-1a, mBAX, and hBAX expression in parent U-251 MG cells and HRE-Bax Clone D cells following 4–24 hours of (a) anoxic (N2) or oxic (O2) treatments; (b) 0.3 or 1% O2 treatments.

Article Snippet: Detection of protein on the immunoblots was performed using anti-human HIF-1a antibody raised in mouse (1:250 dilution; BD Biosciences Clontech, Palo Alto, CA), followed by peroxidaseconjugated horse anti-mouse IgG (1:30,000 dilution; Vector Laboratories, Burlingame, CA), as well as antimurine BAX monoclonal antibody and anti-human BAX monoclonal antibody (1:5000 dilution; R&D System, Inc., Minneapolis, MN) followed by goat anti-mouse IgG (1:50,000 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Techniques: Expressing

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