rabbit anti activating transcription factor 4  (Boster Bio)

Journal: Antioxidants

Article Title: Mitochondrial Unfolded Protein Response (mtUPR) Activation Improves Pathological Alterations in Cellular Models of Ethylmalonic Encephalopathy

doi: 10.3390/antiox14060741

Figure Lengend Snippet: Effect of CoC3 treatment for seven days on the expression levels of proteins of the transcriptional canonical mtUPR axis. ( A ) Cellular extracts from untreated and treated with CoC3 control (C1 and C2) and fibroblasts derived from the EE patient (P) fibroblasts were analysed by Western blot analysis. The membranes were immunostained using antibodies against eif2α, P-eif2α, ATF4, ATF5, HSP60, HSP70, and β-actin, the latter used as a loading control. ( B ) Densitometry of Western blotting. Untreated and treated control samples were unified in one value for each condition (C and C + CoC3, respectively) which represents the mean of each control measurement. Densitometry was referred to the untreated control (C) value. Data were referred to the control and represent the mean ± SD of 3 independent experiments. ** p < 0.01 and *** p < 0.001 between control and patient fibroblasts. aa p < 0.01, and aaaa p < 0.0001 between untreated and treated patient fibroblasts. a.u.: arbitrary units.

Article Snippet: Anti-eukaryotic translation initiation factor 2α (eif2α) (9722S), anti-phospho-eif2α (9721S), anti-mitochondrially encoded NADH Dehydrogenase Subunit 1 (mt-ND1) (6888S), anti-mitochondrial transcription factor A (TFAM) (7495S), and anti-Activating Transcription Factor 4 (ATF4) (11815S) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Expressing, Control, Derivative Assay, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Nanoparticle-Mediated Delivery of 2-Deoxy-D-Glucose Induces Antitumor Immunity and Cytotoxicity in Liver Tumors in Mice

doi: 10.1016/j.jcmgh.2020.10.010

Figure Lengend Snippet: Effects of 2DG or 2DG-PLGA-NPs on ATP production, cytotoxicity, tumor vessel formation, and ROS production. ( A and B ) Total, glycolytic, and mitochondrial ATP production rates were measured in the Huh7 cells (3 × 10 4 cells/well) using a Seahorse XF24 Extracellular Flux Analyzer. The extracellular acidification rate and oxygen consumption rate (OCR) were analyzed after injection of 2DG (0, 1, 5, 10, and 50 mmol/L). Glycolytic and mitochondrial ATP production rates were determined based on the extracellular acidification rates and OCRs using an Agilent Seahorse XFp Real-Time ATP rate assay report generator. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. ( C ) Immunoblots showing results for p-AMPK, AMPK, p-mTOR, mTOR, p-S6, S6, p-Rb, Rb, spliced x-box binding protein 1 (XBP-1s), CCAAT/enhancer binding protein-homologous protein (CHOP), activating transcription factor 4 (ATF4), cyclin D1, proliferating cell nuclear antigen (PCNA), and vascular endothelial growth factor (VEGF)-A, performed using xenograft tumors in each nude mouse group (n = 3). Column a, 2DG (100 mg/kg); column b, 2DG (1000 mg/kg); column c, 2DG-PLGA-NP (80 mg/kg); and column d, 2DG-PLGA-NP (800 mg/kg). Administration frequency was daily for 2DG (intraperitoneal) and weekly for 2DG-PLGA-NPs (intravenous). The amount of phosphorylated protein was normalized to total protein. The amount of some proteins was normalized to that of β-actin. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. ( D ) Immunostained CD34-positive vessels and ( E ) dihydroethidium staining for detecting ROS production in the Huh7 cell xenograft tumors 21 days after the commencement of treatment in the 5 groups as indicated (n = 3 for each group) (original magnification: ×400). Administration frequency was daily for 2DG (intraperitoneal) and weekly for 2DG-PLGA-NPs (intravenous). National Institutes of Health image analysis software was used to quantify the mean percentage of the positively stained area of 5 randomly selected fields in the digital images of each tumor. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. Column 1, control; column 2, 2DG (100 mg/kg); column 3, 2DG (1000 mg/kg); column 4, 2DG-PLGA-NP (80 mg/kg); and column 5, 2DG-PLGA-NP (800 mg/kg). Cont, control; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone.

Article Snippet: The samples subsequently were incubated overnight at 4°C with the following antibodies: a rabbit anti–phospho-AMPKα (Thr172) monoclonal antibody (Cell Signaling Technology), a rabbit anti-AMPKα monoclonal antibody (Cell Signaling Technology), a rabbit anti–phospho-mTOR (Ser2448) monoclonal antibody (Cell Signaling Technology), a rabbit anti-mTOR monoclonal antibody (Cell Signaling Technology), a rabbit anti–phospho-S6 ribosomal protein (Ser235/236) monoclonal antibody (Cell Signaling Technology), a mouse anti-S6 ribosomal protein monoclonal antibody (Cell Signaling Technology), a rabbit anti–phospho-Rb (Ser780) polyclonal antibody (Cell Signaling Technology), a mouse anti-Rb monoclonal antibody (Cell Signaling Technology), a rabbit anti–spliced x-box binding protein 1 monoclonal antibody (Cell Signaling Technology), a mouse anti–CCAAT/enhancer binding protein-homologous protein monoclonal antibody (Cell Signaling Technology), a rabbit anti– activating transcription factor 4 monoclonal antibody (Cell Signaling Technology), a rabbit anti–cyclin D1 monoclonal antibody (Cell Signaling Technology), a mouse anti–proliferating cell nuclear antigen monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit anti–vascular endothelial growth factor polyclonal antibody (Abcam, Cambridge, UK), a rabbit anti–phospho-JAK1 monoclonal antibody (Cell Signaling Technology), a rabbit anti-JAK1 monoclonal antibody (Cell Signaling Technology), a rabbit anti–phospho-JAK2 monoclonal antibody (Cell Signaling Technology), a rabbit anti-JAK2 monoclonal antibody (Cell Signaling Technology), a rabbit anti–phospho-STAT1 (Tyr701) monoclonal antibody (Cell Signaling Technology), a rabbit anti-STAT1 monoclonal antibody (Cell Signaling Technology), a rabbit anti–phospho-EZH2 (Thr311) polyclonal antibody (Cell Signaling Technology), a rabbit anti-EZH2 monoclonal antibody (Cell Signaling Technology), and a rabbit anti–trimethyl-histone H3 (Lys27) polyclonal antibody (Sigma-Aldrich).

Techniques: Injection, Western Blot, Binding Assay, Staining, Software

Journal: Heliyon

Article Title: Melatonin attenuates spatial learning and memory dysfunction in developing rats by suppressing isoflurane-induced endoplasmic reticulum stress via the SIRT1/Mfn2/PERK signaling pathway

doi: 10.1016/j.heliyon.2022.e10326

Figure Lengend Snippet: Melatonin ameliorated isoflurane-induced reduction of SIRT1 and Mfn2 and ER stress in hippocampus of new born rats. (A) Western blot bands of SIRT1 and Mfn2 in the hippocampus. (B) Hippocampal SIRT1 expression decreased after isoflurane exposure. Administration of melatonin significantly attenuated isoflurane-induced decrease in hippocampal SIRT1 expression in neonatal rats ( F (3, 20) = 21.830, P < 0.001). (C) Hippocampal Mfn2 expression decreased after isoflurane exposure. Administration of melatonin significantly attenuated isoflurane-induced decrease in hippocampal Mfn2 expression in neonatal rats ( F (3, 20) = 122.900, P < 0.001). (D) Western blot bands of endoplasmic reticulum stress signaling in the hippocampus. (E) Administration of melatonin significantly reduced isoflurane induced increase of endoplasmic reticulum stress marker protein CHOP expression in hippocampus of neonatal rats ( F (3, 20) = 28.890, P < 0.001). (F) Administration of melatonin significantly reduced isoflurane induced increase of endoplasmic reticulum stress marker protein PERK expression in hippocampus of neonatal rats ( F (3, 20) = 18.690, P < 0.001). (G) Administration of melatonin significantly reduced isoflurane induced increase of endoplasmic reticulum stress marker protein caspase 12 expression in hippocampus of neonatal rats ( F (3, 20) = 26.990, P < 0.001). (H) Administration of melatonin significantly reduced isoflurane induced increase of endoplasmic reticulum stress marker protein GRP78 expression in hippocampus of neonatal rats ( F (3, 20) = 26.010, P < 0.001). (I) Administration of melatonin significantly reduced isoflurane induced increase of endoplasmic reticulum stress marker protein ATF4 expression in hippocampus of neonatal rats ( F (3, 20) = 32.240, P < 0.001). Data were presented as mean ± SEM (n = 6). ∗denotes p < 0.05, ∗∗denotes p < 0.01, ∗∗∗denotes p < 0.001 when comparing the two groups under each end of the capped line.

Article Snippet: The primary antibodies included the following: mouse anti-β-actin (1:2000; Qidongzi, Wuhan, China), mouse anti-SIRT1 (1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-mitofusin 2 (Mfn2) (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-protein kinase RNA-like ER kinase (PERK) (1:500; Affinity Biosciences, OH, USA), rabbit anti- C/EBP homologues protein (CHOP) (1:500; Affinity Biosciences, OH, USA), rabbit anti-78 kDa glucose regulated protein (GRP78) (1:500; Affinity Biosciences, OH, USA), rabbit anti-activating transcription factor 4 (ATF4) (1:500; Affinity Biosciences, OH, USA), rabbit anti-cysteine containing aspartate specific protease 12 (caspase 12) (1:500; Proteintech North America, IL, USA), rabbit anti-Bcl-2-associated X protein (Bax) (1:500; Proteintech North America, IL, USA), rabbit anti-post-synaptic density protein 95 (PSD95) (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-B-cell lymphoma-2 (Bcl-2) (1:500; Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-cleaved-cysteine containing aspartate specific protease 3 (cleaved-caspase 3).

Techniques: Western Blot, Expressing, Marker