atf 6  (Cell Signaling Technology Inc)

Journal: ACS Biomaterials Science & Engineering

Article Title: Self-Assembled Quercetin–Mecobalamin Nanoparticles Mitigate Ischemic Stroke Injury via Enhanced Neuroprotection and Microglial Modulation

doi: 10.1021/acsbiomaterials.5c01855

Figure Lengend Snippet: Qu-Me NPs attenuates MCAO-induced injury. (A,B) Open-field test performed 24 h post-MCAO shows improved locomotor activity in Qu- and Qu-Me NPs-treated mice compared with the MCAO and Me groups ( n = 6). (C,D) Y-maze test demonstrates ameliorated cognitive function in the Qu- and Qu-Me NPs-treated mice compared with the MCAO and Me groups ( n = 6). (E) Modified neurological severity scores (mNSS) for each group (Sham, MCAO, Me, Qu, and Qu-Me NPs) evaluated at 24 h post-MCAO ( n = 6). (F–J) Western blot analysis illustrates reduced expression of ER stress markers (ATF-6, CHOP) and pro-inflammatory cytokines (IL-1β, IL-6) in the Qu and Qu-Me NP groups compared with MCAO ( n = 3). Results are presented as averages ±SD * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. Analysis was performed using one-way ANOVA followed by Dunnett multiple-comparisons test for mean differences.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and incubated overnight with specific primary antibodies against β-actin (1/5000, ab170325, Abcam), ATF-6 (1:1000, A0202, ABclonal), CHOP (1:1000, A21902 , ABclonal), IL-1β (1/1000, 16806-1-AP, Proteintech), IL-6, pannexin1 (1/1000, ab124969, Abcam), and DDIT4 (1:1000, ab191871, Abcam).

Techniques: Activity Assay, Modification, Western Blot, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: USP19 promotes cytoplasmic mislocalization of TDP-43 and enhances ER stress. ( A ) Representative images of TDP-43-tomato (red), USP19 (green), and DAPI (blue) in HeLa cells transfected with TDP-43 and control siRNA or USP19 siRNA ± proteasome inhibition with MG132 treatment (10 μM, 4 h). ( B ) Quantification of TDP-43-tomato in the cytoplasm vs. nucleus with MG132 treatment (one-sample t test, * P < 0.05; n = 3 independent experiments with 10 to 32 cells/condition averaged per experiment). ( C ) Representative images of TDP-43-tomato (red), USP19 (green), and DAPI (blue) in HeLa cells transfected with TDP-43 and USP19 variants or GFP control. ( D ) Quantification of TDP-43-tomato in the cytoplasm vs. nucleus (one-way ANOVA, F(2, 6) = 22.73, P = 0.0016; post hoc Dunnett, ** P < 0.005, n = 3 independent experiments with 13 to 33 cells/condition averaged per experiment). ( E ) Representative images of endogenous USP19–TDP-43 PLA puncta (green) and DAPI (blue) in HeLa cells transfected with control or USP19 siRNA. Negative controls with only 1 primary antibody or 1 secondary antibody probe ( Bottom ). ( F ) Representative images of endogenous USP19–TDP-43 PLA puncta (red) and DAPI (blue) in HeLa cells treated ± ER stressor tunicamycin (2 μM) for 24 h. Negative controls with only 1 primary antibody or 1 secondary antibody probe ( Bottom ). ( G ) Quantification of USP19–TDP-43 PLA intensity ± tunicamycin treatment (2 μM, 24 h) (one-sample t test, *** P < 0.001, n = 3 independent experiments with 29 to 36 cells/condition averaged per experiment). ( H ) Schematic of unfolded protein response (UPR) triggered by ER stressors, such as misfolded proteins, illustrating the activation of IRE1α-XBP1, PERK-ATF4, and ATF6 pathways, leading to CHOP induction. ( I ) Representative blots of ER stress-induced UPR markers from tet-induced HeLa-GFP-TDP-43 cells transfected with control or USP19 siRNA and treated ± tunicamycin (2 μM; 0, 8, 24 h). ( J – M ) Quantification of CHOP, cleaved ATF6 (cl-ATF6), ATF4, and IRE1α (two-way ANOVA; CHOP: F(1, 18) = 24.49, P = 0.0001; cl-ATF6: F(1, 18) = 41.93, P < 0.0001; ATF-4: F(1, 18) = 4.343, P = 0.0517; IRE1α: F(1, 18) = 2.80, P = 0.1571; post hoc Sidak, * P < 0.05, ** P < 0.005, *** P < 0.001, ns: not significant; n = 4 independent experiments with 2 to 3 replicates averaged per experiment).

Article Snippet: Primary antibodies used in Western blotting: Ubiquitin-K48 (Abcam Cat# ab140601), Ubiquitin-K63 (Abcam, Cat# ab179434), Myc (Cell signaling, #2276s), Flag (MA5-48063), Actin (Santa Cruz, sc-47778), TDP-43 (Cell signaling, #3448), Tubulin (Cell signaling, #2146); USP19 (Abcam, #93159), NUP98 (cell signaling, #2598s), ATF4 (Cell signaling, #11815s), ATF6 (Cell signaling, #65880S), CHOP (Cell signaling, #2895s), IRE1α (Cell signaling, #3294s), HA (Cell signaling, 2367s).

Techniques: Transfection, Control, Inhibition, Activation Assay