rabbit polyclonal against lc3 catalog no. pd014 antibody  (MBL International)

Journal: Cell Death & Disease

Article Title: Sphingosine kinase 1-associated autophagy differs between neurons and astrocytes

doi: 10.1038/s41419-018-0599-5

Figure Lengend Snippet: a Cultured primary cortical neurons were treated with a vehicle (control, cont), or with 10-NCP (10-NCP, 5 µM), or fluphenazine (FPZ, 5 µM) for 4 h. Lysates were analyzed by western blotting with antibodies against LC3. Actin was used as a loading control. Bar graphs represent the quantification of the LC3-II intensities normalized to actin. * p (cont vs 10-NCP) = 0.042, * p (cont vs FPZ) = 0.024. b Cultured primary astrocytes were treated with a vehicle (control, cont), or with 10-NCP (10-NCP, 5 µM), or fluphenazine (FPZ, 5 µM), with or without NH 4 Cl for 4 h. Lysates were analyzed by western blotting with antibodies against LC3. Actin was used as a loading control. Bar graphs represent the quantification of the LC3-II intensities normalized to actin. p LC3 (cont vs 10-NCP) = 0.0749, p LC3 (cont vs FPZ) = 0.5297 (one-way ANOVA). *** p (cont vs cont + NH 4 Cl) = 0.0002 ( t -student), p (cont + NH 4 Cl vs 10-NCP + NH 4 Cl) = 0.7699, p (cont + NH 4 Cl vs FPZ + NH 4 Cl) = 0.2257 (one-way ANOVA). Results were pooled from three independent experiments. c , d Cultured primary cortical neurons ( c ) and primary astrocytes ( d ) were treated with a vehicle (control, cont) or with 10-NCP (5 µM), or fluphenazine (FPZ, 5 µM) for 4 h. Lysates were analyzed by western blotting with antibodies against phosphorylated SK1 (pSK1) or pan-SK1 (SK1). Actin was used as a loading control. Bar graphs represent the quantification of the pSK1 and SK1 band intensities normalized to actin. In neurons: * p pSK1 (cont vs 10-NCP) = 0.0269, * p pSK1 (cont vs FPZ) = 0.0224; p SK1 (cont vs 10-NCP) = 0.7939, p SK1 (cont vs FPZ) = 0.208 (one-way ANOVA). In astrocytes: p pSK1 (cont vs 10-NCP) = 0.05356, p pSK1 (cont vs FPZ) = 0.9909; p SK1 (cont vs 10-NCP) = 0.1158, p SK1 (cont vs FPZ) = 0.4113 (one-way ANOVA). Results were pooled from three independent experiments. e Primary astrocytes were transfected with GFP and RFP-LC3 or with SK1-GFP and RFP-LC3. Twenty-four hours after transfection, astrocytes were imaged. Scale bar is 10 µm. A part of the image is zoomed in to visualize the SK1-GFP and RFP-LC3 puncta. f Quantification of the RFP-LC3 puncta index from e . *** p (GFP vs SK1-GFP) = 0.0001 ( t test). One hundred and fifty cells were analyzed from three independent experiments. g The photoswitchable protein Dendra2 targeted to LC3 as a surrogate for the flux through autophagy. Brief irradiation with short-wave-length visible light causes Dendra2-LC3 to undergo an irreversible conformational change (“photoswitch”) and emit red fluorescence that can be tracked until altered Dendra2-LC3 is cleared. A cell is outlined to show cellular morphology. Scale bar is 10 µm. h Primary astrocytes were transfected with Dendra2-LC3 and an empty plasmid or with Dendra2-LC3 and a plasmid that encodes Beclin1, or Dendra2-LC3 and SK1. After the photoswitch, astrocytes were longitudinally imaged. The change in red fluorescence intensity over time was used to calculate the half-life of Dendra2-LC3. The single-cell half-life of Dendra2-LC3 was reduced by SK1 expression. Beclin1 was used as a positive control . Fifty astrocytes were analyzed per condition from two independent experiments. *** p (control vs Becl) = 0.0001, *** p (control vs SK1) = 0.0001; p (Becl vs SK1) = 0.084 (one-way ANOVA). i , j Cultured primary astrocytes were maintained in DMEM supplemented with 10% fetal bovine serum (control, cont) or in Hanks’ balanced salt solution (starvation, starv) for 4 h. Samples were also treated with 10 mM NH 4 Cl to block the last step of autophagy-mediated degradation. Lysates were analyzed by western blotting with antibodies against LC3 ( i ) or with antibodies against phosphorylated SK1 (pSK1) or pan-SK1 (SK1) ( j ). Actin was used as a loading control. Bar graphs represent the quantification of the LC3-II, pSK1, or SK1 intensities normalized to actin. * p LC3 (control vs starv) = 0.017; p pSK1 (control vs starv) = 0.0072, p SK1 (control vs starv) = 0.331 (one-way ANOVA). Results were pooled from three independent experiments. n.s. not significant

Article Snippet: Antibodies against LC3 were from MBL (#PD014, 1:1000; Woburn, MA).

Techniques: Cell Culture, Control, Western Blot, Transfection, Irradiation, Fluorescence, Plasmid Preparation, Expressing, Positive Control, Blocking Assay

Journal: Cell Death & Disease

Article Title: Sphingosine kinase 1-associated autophagy differs between neurons and astrocytes

doi: 10.1038/s41419-018-0599-5

Figure Lengend Snippet: a Three cohorts of primary neurons were transfected with RFP-LC3, GFP, and an empty plasmid. The fourth cohort of neurons was transfected with RFP-LC3, GFP, and a plasmid that encodes SK1. The first three cohorts of transfected neurons were treated with a vehicle (control), or with 0.5 µM 10-NCP or with 5 µM 10-NCP for 4 h, respectively. Neurons were imaged 24 h after transfection. Scale bar is 20 µm. b Quantification of the RFP-LC3 puncta index from a . SK1-expressed neurons and neurons treated with 5 µM 10-NCP exhibit higher RFP-LC3 puncta index compared to control (cont). *** p (cont vs 5 µM 10-NCP) = 0.0001, ** p (cont vs SK1) = 0.0019, * p (cont vs 0.5 µM 10-NCP) = 0.0342 (one-way ANOVA). Two hundred neurons were analyzed from three independent experiments. c The graph represents a correlation between RFP-LC3 puncta index from b and risk of death of four cohorts of neurons (neurons transfected with RFP-LC3, GFP, and an empty plasmid, and treated with a vehicle; neurons transfected with RFP-LC3, GFP, and a plasmid that encodes SK1; neurons transfected with RFP-LC3, GFP, and an empty plasmid, and treated with 0.5 µM 10-NCP for 4 h; and neurons transfected with RFP-LC3, GFP, and an empty plasmid, and treated with 5 µM 10-NCP for 4 h. *** p (cont vs 5 µM 10-NCP) = 0.0001, *** p (cont vs SK1) = 0.0001; p (cont vs 0.5 µM 10-NCP) = 0.352 (log-rank test). Two hundred neurons were analyzed from three independent experiments. d Four cohorts of primary cortical neurons were cultured. Three cohorts of neurons were nucleofected with GFP. The first cohort was treated with a vehicle (control, cont). The second and the third cohorts were treated with 0.5 or 5 µM 10-NCP for 4 h. The fourth cohort was nucleofected with SK1-GFP (SK1). Neurons were plated and maintained for 36 h. Neurons were then collected, and the levels of S1P were measured by liquid chromatography and mass spectrometry. The bar graph represents the S1P levels normalized with S1P content in the control group. *** p (cont vs SK1) = 0.0001, * p (cont vs 0.5 µM 10-NCP) = 0.0274, *** p (cont vs 5 µM 10-NCP) = 0.0012 (one-way ANOVA). Results were pooled from three independent experiments. A.u. arbitrary units, n.s. not significant

Article Snippet: Antibodies against LC3 were from MBL (#PD014, 1:1000; Woburn, MA).

Techniques: Transfection, Plasmid Preparation, Control, Cell Culture, Liquid Chromatography, Mass Spectrometry