Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Effects on Autophagy of Moxibustion at Governor Vessel Acupoints in APP/PS1double-Transgenic Alzheimer's Disease Mice through the lncRNA Six3os1/miR-511-3p/AKT3 Molecular Axis

doi: 10.1155/2022/3881962

Figure Lengend Snippet: LC3B-II/I ratio and P62 hippocampal expression (mean ± SD, n = 6). Note: ∗∗ indicates significant difference versus the control group, at p < 0.01; ## indicates significant difference versus the AD model group, at p < 0.01.

Article Snippet: PI3K (ab86714, Abcam), AKT3 (bs-5146R, Bioss), mTOR (2972s, CST), LC3B (bs-2912R, Bioss), P62 (18420-1-AP, Triple Eagle), western removal buffer of primary antibody and second antibody (P0025, Beyotime), RIPA cell lysate (P0013B, Beyotime), ECL hypersensitive luminescence kit (34095, Thermo), goat anti-mouse IgG secondary antibody (ZB-2305, Zsbio), goat anti-rabbit IgG secondary antibody (ZB-2301, Zsbio), goat anti-rabbit IgG (FITC) (B029, Ebiogo), sheep serum block (B010, Ebiogo), anti-fluorescence quench blocking agent (containing DAPI) (B024, Ebiogo), TRIzol (15596026, Life Technologies), and hematoxylin (BA-4041, BaSO).

Techniques: Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Effects on Autophagy of Moxibustion at Governor Vessel Acupoints in APP/PS1double-Transgenic Alzheimer's Disease Mice through the lncRNA Six3os1/miR-511-3p/AKT3 Molecular Axis

doi: 10.1155/2022/3881962

Figure Lengend Snippet: Hippocampal expression of LC3B and P62 (mean ± SD, n = 3). Note: Nuclei are in blue and LC3B/P62 are in green. (a) LC3B average optical density; (b) P62 average optical density. ∗∗ indicates significant difference versus the control group, at p < 0.01; ## indicates significant difference versus the AD model group, at p < 0.01.

Article Snippet: PI3K (ab86714, Abcam), AKT3 (bs-5146R, Bioss), mTOR (2972s, CST), LC3B (bs-2912R, Bioss), P62 (18420-1-AP, Triple Eagle), western removal buffer of primary antibody and second antibody (P0025, Beyotime), RIPA cell lysate (P0013B, Beyotime), ECL hypersensitive luminescence kit (34095, Thermo), goat anti-mouse IgG secondary antibody (ZB-2305, Zsbio), goat anti-rabbit IgG secondary antibody (ZB-2301, Zsbio), goat anti-rabbit IgG (FITC) (B029, Ebiogo), sheep serum block (B010, Ebiogo), anti-fluorescence quench blocking agent (containing DAPI) (B024, Ebiogo), TRIzol (15596026, Life Technologies), and hematoxylin (BA-4041, BaSO).

Techniques: Expressing

Journal: Neural Regeneration Research

Article Title: Apelin-13 inhibits apoptosis and excessive autophagy in cerebral ischemia/reperfusion injury

doi: 10.4103/1673-5374.300725

Figure Lengend Snippet: Apelin-13 alleviates autophagy in cerebral ischemia/reperfusion models in vivo and in vitro . (A) Double immunofluorescent staining of LC3B (green, stained by fluorescein isothiocyanate) and NeuN (red, stained by Cy5) in the rat hippocampus. Apelin-13 treatment significantly decreased the expression of LC3B in the hippocampus of MCAO rats. (B) Representative western blots and quantitative evaluations of LC3B in the rat hippocampus. (C) Double immunofluorescent staining of p62 (green, stained by fluorescein isothiocyanate) and NeuN (red, stained by Cy5) in the rat hippocampus. Apelin-13 treatment significantly increased the expression of p62 in the hippocampus of MCAO rats. (D) Representative western blots and quantitative evaluations of p62 in the rat hippocampus. (E) Acridine orange staining of SH-SY5Y cells. Arrows indicate autophagic vacuoles. Scale bars: 500 μm in A and C, 100 μm in E. (F, G) Representative western blots and quantitative evaluations of LC3B and p62 in SH-SY5Y cells. Data are represented as the mean ± standard error of the mean ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . sham or control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . MCAO or OGD/R group (one-way analysis of variance followed by post hoc Tukey’s test). DAPI: 4’,6-Diamidino-2-phenylindole; LC3B: microtubule-associated protein 1 light chain 3; MCAO: middle cerebral artery occlusion; OD: optical density; OGD/R: oxygen-glucose deprivation/reperfusion.

Article Snippet: After blocking at room temperature for 1.5 hours with 5% non-fat milk powder in Tris-buffered saline with Tween-20, the membranes were incubated at 4°C overnight with the following primary antibodies against autophagy and apoptosis markers and other relevant pathway proteins: mouse anti-Bcl-2 (1:1000; Cat# 15071; Cell Signaling Technology), rabbit anti-Bax (1:1000; Cat# 2772; Cell Signaling Technology), rabbit anti-phospho(p)-Akt (1:1000; Cat# 4060; Cell Signaling Technology), mouse anti-Akt (1:1000; Cat# 2920; Cell Signaling Technology), rabbit anti-mTOR (1:1000; Cat# 2983; Cell Signaling Technology), rabbit anti-p-mTOR (1:1000; Cat# ab84400; Cell Signaling Technology), rabbit anti-PI3K (1:1000; Cat# 4292; Cell Signaling Technology), rabbit anti-p-PI3K (1:1000; Bioworld, Nanjing, China), mouse anti-p62 (1:1000; Cat# ab91526; Abcam), rabbit anti-Beclin1 (1:1000; Cat# ab62557; Abcam), rabbit anti-caspase-3 (1:1000; Cat# BS4605; Proteintech, Wuhan, China), rabbit anti-LC3B (1:1000; Cat# NB100-2220; NOVUS), and mouse anti-β-actin (1:2500; Cat# TA-09; Zhongshan Golden Bridge Biotechnology, Beijing China).

Techniques: In Vivo, In Vitro, Staining, Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Apelin-13 inhibits apoptosis and excessive autophagy in cerebral ischemia/reperfusion injury

doi: 10.4103/1673-5374.300725

Figure Lengend Snippet: LY294002 or rapamycin treatment enhances apoptosis and autophagy in vitro . (A–C) Representative western blots and quantitative evaluations of LC3B (A), p62 (B), and the ratio of Bcl-2/Bax (C) in SH-SY5Y cells. Data are represented as the mean ± standard error of the mean ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . OGD/R group; ++ P < 0.01, +++ P < 0.001, vs . OGD/R + apelin-13 group (one-way analysis of variance followed by post hoc Tukey’s test). LY294002 and rapamycin are inhibitors of the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin pathway. LC3B: Microtubule-associated protein 1 light chain 3; OGD/R: oxygen-glucose deprivation/reperfusion.

Article Snippet: After blocking at room temperature for 1.5 hours with 5% non-fat milk powder in Tris-buffered saline with Tween-20, the membranes were incubated at 4°C overnight with the following primary antibodies against autophagy and apoptosis markers and other relevant pathway proteins: mouse anti-Bcl-2 (1:1000; Cat# 15071; Cell Signaling Technology), rabbit anti-Bax (1:1000; Cat# 2772; Cell Signaling Technology), rabbit anti-phospho(p)-Akt (1:1000; Cat# 4060; Cell Signaling Technology), mouse anti-Akt (1:1000; Cat# 2920; Cell Signaling Technology), rabbit anti-mTOR (1:1000; Cat# 2983; Cell Signaling Technology), rabbit anti-p-mTOR (1:1000; Cat# ab84400; Cell Signaling Technology), rabbit anti-PI3K (1:1000; Cat# 4292; Cell Signaling Technology), rabbit anti-p-PI3K (1:1000; Bioworld, Nanjing, China), mouse anti-p62 (1:1000; Cat# ab91526; Abcam), rabbit anti-Beclin1 (1:1000; Cat# ab62557; Abcam), rabbit anti-caspase-3 (1:1000; Cat# BS4605; Proteintech, Wuhan, China), rabbit anti-LC3B (1:1000; Cat# NB100-2220; NOVUS), and mouse anti-β-actin (1:2500; Cat# TA-09; Zhongshan Golden Bridge Biotechnology, Beijing China).

Techniques: In Vitro, Western Blot

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2

doi: 10.1038/s41374-019-0327-5

Figure Lengend Snippet: CB1 knockout elevated autophagy in the liver of HBs transgenic mice. a Representative immunohistochemical analysis of paraffin-embedded liver sections from 52-week-old mice was performed using anti-LC3B antibody. Original image magnification ×1000, bar 40 µm. b Quantification of LC3B puncta assessed with ImageJ software and expressed as % of LC3B staining/field. c Western blot analysis of lysates from 52-week-old mice was performed using specific anti-LC3B and anti-p62 antibodies. d Representative immunofluorescence analysis of paraffin-embedded liver sections of 52-week-old mice was performed using specific anti-LAMP1 antibodies (red). Nuclei were stained with DAPI (blue). Magnification ×1000, bar 40 µm

Article Snippet: Proteins recognition was done by using specific antibodies against AMPK (Genetex, GTX50863-100), Phospho-AMPK (Genetex, GTX130429-25), LC3B (Novus, NB100-2220), p62 (Proteintech, 18420-1-AP), PLIN2 (Proteintech, 15294-1-AP), PLIN4 (Merck, ABS526), PLIN3, PLIN5 (Progen: GP30S; GP31S), GAPDH (Proteintech,60004-1-Ig), and β actin (Santa Cruz, sc47778).

Techniques: Knock-Out, Transgenic Assay, Immunohistochemical staining, Software, Staining, Western Blot, Immunofluorescence

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2

doi: 10.1038/s41374-019-0327-5

Figure Lengend Snippet: a Double immunofluorescence analysis of paraffin-embedded liver sections of 52-week-old mice was performed using specific anti-LC3B (green) and LAMP1 (red) antibodies. Nuclei were stained with DAPI (blue). Colocalization of these two proteins appears in yellow. Magnification ×1000 Scale bar 40 µm. b Representative double immunofluorescence analysis of paraffin-embedded liver sections from 52-week-old mice was performed using anti-p62 (green) and anti-LAMP1 (red). Arrowheads indicate colocalization of these proteins shown in yellow color and p62 accumulation in nuclear inclusion. Original image magnification ×1000, bars 40 µm. c Representative immunohistochemical analysis of paraffin-embedded liver sections from 52-week-old mice was performed using specific anti-LAL (lysosomal acid lipase) antibody. Original magnification ×200 and ×1000, bar 100 µm. d Representative immunohistochemical analysis of PLIN2 (blue) and LAMP1 (red). Arrows show costained areas (purple color). Original magnification ×200 and ×1000, bar 100 µm

Article Snippet: Proteins recognition was done by using specific antibodies against AMPK (Genetex, GTX50863-100), Phospho-AMPK (Genetex, GTX130429-25), LC3B (Novus, NB100-2220), p62 (Proteintech, 18420-1-AP), PLIN2 (Proteintech, 15294-1-AP), PLIN4 (Merck, ABS526), PLIN3, PLIN5 (Progen: GP30S; GP31S), GAPDH (Proteintech,60004-1-Ig), and β actin (Santa Cruz, sc47778).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining

Journal: Neural Regeneration Research

Article Title: GIT1 enhances neurite outgrowth by stimulating microtubule assembly

doi: 10.4103/1673-5374.179054

Figure Lengend Snippet: GIT1 interacted with tubulins and microtubule-associated proteins, and stimulated microtubule assembly. (A) Brain extract from 8-week-old mice was immunoprecipitated with the GIT1 antibody, and western blots were probed by antibodies against CRMP2, Clasp2, α-tubulin, Tuj1, TUBGCP3, MAP2, Tau, MAP1B and GIT1. The images show the colocalization of Tuj1 (red) and GIT1 (green) (B), Clasp2 (red) and GIT1 (green) (C), and MAP2 (red) and GIT1 (green) (D) in primary cultured hippocampal neurons on postnatal day (P) 0. Scale bars: 20 μm. (E) Expression levels of Tuj1, MAP2 and GIT1 in the cortex and hippocampus of wild-type (WT) and GIT1 heterozygote (HET) mice on postnatal day (P) 0, P15 and P30. (F) GIT1 activates tubulin polymerization in vitro . Result represents means of three independent observations. min: Minutes.

Article Snippet: After blocking in milk, the blots were incubated in primary antibody against GIT1 (goat, 1:800; Santa Cruz Biotechnology), Clasp2 (rabbit, 1:600; Biorbyt), CRMP2 (rabbit, 1:1,000; Proteintech, Chicago IL, USA), MAP2 (rabbit, 1:1,000; Cell Signaling Technology), Tuj1 (rabbit, 1:1,000; Sigma-Aldrich), TUBGCP3 (rabbit, 1:1,000; Proteintech), MAP1B (rabbit, 1:1,000; Proteintech), Tau (rabbit, 1:1,000; Sigma-Aldrich) or beta-actin (mouse, 1:2,000; Millipore, Darmstadt, Germany) at 4°C overnight.

Techniques: Immunoprecipitation, Western Blot, Cell Culture, Expressing, In Vitro

Journal: Scientific Reports

Article Title: 3β, 6β-dichloro-5-hydroxy-5α-cholestane facilitates neuronal development through modulating TrkA signaling regulated proteins in primary hippocampal neuron

doi: 10.1038/s41598-019-55364-8

Figure Lengend Snippet: List of differentially expressed proteins in primary culture of hippocampal neurons treated with SCH vs vehicle.

Article Snippet: The following antibodies were used for immunostaining: primary antibodies to tubulin α-subunit (mouse monoclonal 12G10, 1:1000 dilution; Developmental Studies Hybridoma Bank, University of Iowa, IO), ankyrin G (rabbit polyclonal H-215, 1:50 dilution; Santa Cruz Biotechnology Inc., CA), MAP2 (mouse monoclonal clone HM-2; 1:500; Sigma, MO), Tau (rabbit polyclonal LF-PA0172; 1:500; Abfrontier, Seoul, Korea), Hnrnpa2b1 (Goat polyclonal, 1:500; Santa Cruz Biotechnology Inc., CA), Map1b (rabbit polyclonal; 1:500; Cell Signaling Technology, Danvers, MA), and secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG [1:1,000], Alexa Fluor 568-conjugated goat anti-rabbit IgG [1:1,000], Alexa Fluor 568-conjugated rabbit anti-goat IgG [1:1,000], Molecular Probes, OR).

Techniques: Expressing, Binding Assay