Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Manganese activates autophagy and microglia M2 polarization against endoplasmic reticulum stress-induced neuroinflammation: Involvement of GSK-3β signaling.

doi: 10.1016/j.biopha.2023.116053

Figure Lengend Snippet: Fig. 5. The effect of pretreatment with 4-PBA or 3-MA on the Mn-induced autophagy. (A-B) Representative the immunoblots bands of ULK1, p-ULK1, p62, and LC3B among groups in western blot assay, which were semi-quantified and analyzed by ImageJ software and shown in bar graphs. Data are expressed as mean ± SD (n = 4). **P < 0.01, and *P < 0.05 Mn group vs. control. ##P < 0.01 4-PBA+Mn or 3-MA+Mn group vs. Mn group.

Article Snippet: Mouse anti-STAT3 antibodies and rabbit antieIF2a, anti-phospho-eIF2a (Ser51), anti-phospho-ULK1 (Ser757), antiSTAT3 primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Software, Control

Journal: Journal of translational medicine

Article Title: Nicotinamide mononucleotide combined with PJ-34 protects microglial cells from lipopolysaccharide-induced mitochondrial impairment through NMNAT3-PARP1 axis.

doi: 10.1186/s12967-025-06280-1

Figure Lengend Snippet: Fig. 3 The down-regulation of NMNAT3 activates PARP1, inhibits the initiation of autophagy, and leading to mitochondrial impairment. (a) Representa tive immunoblots of NMNAT3, PAR polymer, p-ULK1 and Beclin1 in BV2 cells transfected with shRNA. β-actin was used as a loading control. (b) Quantita tive analysis of NMNAT3 knockdown efficiency (n = 3; +, p < 0.05). (c) Quantification of total PARylation levels in BV2 cells transfected with shRNA (n = 3; #, p < 0.05; ###, p < 0.001; ++, p < 0.01). (d) Quantification of relative expression levels of p-ULK1 protein in BV2 cells transfected with shRNA (n = 3; #, p < 0.05; ##, p < 0.01, ++, p < 0.01). (e) Quantification of relative expression levels of Beclin1 protein in BV2 cells transfected with shRNA (n = 3; #, p < 0.05; +, p < 0.05). (f) Quantification of ATP levels in BV2 cells transfected with shRNA (n = 3; ##p < 0.01; ###p < 0.001, ***p < 0.001). (g) The quantification of the MMP in BV2 cells transfected with shRNA (n = 3; #p < 0.05; +++, p < 0.001) (h) Representative images of JC-1 staining in in BV2 cells transfected with shRNA analyzed by flow cytometry. Data are presented as mean ± SEM. Statistical analyses were performed with unpaired Student’s t-test (b) and multiple t-tests (c-g). Abbre viations: LPS, lipopolysaccharide; NMNAT3, Nicotinamide mononucleotide adenylyl transferase 3; PAR, poly ADP-ribose; MMP, Mitochondrial membrane potential; ULK1, Unc-51-like kinase 1

Article Snippet: Membranes were incubated at 4°C overnight with different primary antibodies: NMNAT3 (1:200, sc-390443, Santa Cruz, CA, USA), PARP1 (1:1,000, DF7198, Affinity, OH, USA), PAR polymer (1:1,000, 4335-MC-100, Trevigen, MD, USA), Beclin1 (1:200, sc-48341, Santa Cruz, CA, USA), p-ULK1 (1:1,000, AF4387, Affinity, OH, USA), ULK1 (1:200, sc-390904, Santa Cruz, CA, USA), LC3 (1:200, sc-398822, Santa Cruz, CA, USA), p62 (1:200, sc-48402, Santa Cruz, CA, USA), Tomm20 (1:1,000, AF5206, Affinity, OH, USA), PINK1 (1:200, sc-518052, Santa Cruz, CA, USA), Parkin (1:200, sc-32282, Santa Cruz, CA, USA), p-Drp1 (Ser616) (1:1,000, AF8470, Affinity, OH, USA), Drp1 (1:1,000, Abcam, ab184247, MA, USA), Mfn2 (1:200, sc-515647, Santa Cruz, CA, USA), Opa1 (1:200, sc-393296, Santa Cruz, CA, USA), p-TBK1(Ser172) (1:1,000, AF8190, Affinity, OH, USA), TBK1 (1:200, sc-52957, Santa Cruz, CA, USA), OPTN (1:200, sc-166576, Santa Cruz, CA, USA), β-actin (1:3,000, AF7018, Affinity, OH, USA), GAPDH (1:1,000, AF7021, Affinity, OH, USA) and Tubulin-β (1:1,000, AF7011, Affinity, OH, USA).

Techniques: Western Blot, Polymer, Transfection, shRNA, Control, Knockdown, Expressing, Staining, Flow Cytometry, Membrane