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

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