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

Article Title: Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

doi: 10.1073/pnas.1524575113

Figure Lengend Snippet: The expression of autophagy markers in the brain and peripheral tissues of adult ubiquitous Htt KO mice. (A–C) Representative immunoblots (A) and quantification of (B) Htt, P62, and (C) LC3I/II ratio in ubiquitous KO and control mice (n = 3 independent experiments). (D and E) Western blotting of the spleen (D) and liver (E) in control and ubiquitous KO mice. The blots were probed by antibodies to cleaved caspase3, P62, LC3I/II, NF-κB, and FAT10 after tamoxifen injection for 3 d. Alpha-tubulin was probed as a loading control. Animal ID numbers are indicated. Ctl: heterozygous floxed Htt/CAG-CreER mice injected with tamoxifen; KO: homozygous floxed Htt/CAG-CreER mice injected with tamoxifen.

Article Snippet: Rabbit antibodies used in this study were obtained from commercial sources as follows: HTT [EPR5526] (Abcam), beta-tubulin III [EP1569Y] (Novus Biologicals), NF-κB [D14E12] (Cell Signaling), LC3I/II [NB100-2220] (Novus Biologicals), ELA3B [GTX105123] (GeneTex), FAT10 [EPR4370] (GeneTex), SPINK3 [2744S] (Cell Signaling), HA [C29F4] (Cell Signaling), p-NF-κB [93H1] (Cell Signaling), RIP3 [GTX107574] (GeneTex), Caspase3 [9662] (Cell Signaling), and cleaved Caspase3 [5A1E] (Cell Signaling).

Techniques: Expressing, Western Blot, Injection

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

Article Title: Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

doi: 10.1073/pnas.1524575113

Figure Lengend Snippet: Normal brains in adult mice that have depleted Htt. (A) The brain photos of 2-mo-old ubiquitous KO mice that had been injected with tamoxifen for 5 d. (B) Brain volumes of 2-mo-old ubiquitous KO mice in A and neuronal KO mice that were injected with tamoxifen at 2 mo of age and used to isolate their brains at the 90th day after tamoxifen injection (one-way ANOVA test, ubiquitous KO: n = 7, P = 0.8759, F = 0.1335; n = 7; neuronal KO: P = 0.9665, F = 0.03416; n.s. represents no significant differences). (Scale bar, 3 mm.) (C and D) Western blotting analysis of the brain tissues from 2-mo-old ubiquitous (C) and neuronal (D) KO mice. Htt, neuronal (NeuN, β-tubulin III), astrocytic (GFAP), autophagic (P62, LC3I/II), apoptotic (cleaved caspase3), and GAPDH were detected by their antibodies. Control is heterozygous floxed Htt/CAG-CreER (C) or heterozygous floxed Htt/Nestin-CreER (D) mice injected with tamoxifen and homozygous floxed Htt/CAG-CreER (C) or homozygous floxed Htt/Nestin-CreER (D) mice injected with corn oil. KO is homozygous floxed Htt/CAG-CreER (C) or homozygous floxed Htt/Nestin-CreER (D) mice injected with tamoxifen. See Fig. 1 for abbreviations key. (E) Immunostaining of GFAP, LC3I/II, and β-tubulin III in the cortex of 2-, 4- and 8-mo-old ubiquitous KO and control mice. (Scale bar, 50 μm.)

Article Snippet: Rabbit antibodies used in this study were obtained from commercial sources as follows: HTT [EPR5526] (Abcam), beta-tubulin III [EP1569Y] (Novus Biologicals), NF-κB [D14E12] (Cell Signaling), LC3I/II [NB100-2220] (Novus Biologicals), ELA3B [GTX105123] (GeneTex), FAT10 [EPR4370] (GeneTex), SPINK3 [2744S] (Cell Signaling), HA [C29F4] (Cell Signaling), p-NF-κB [93H1] (Cell Signaling), RIP3 [GTX107574] (GeneTex), Caspase3 [9662] (Cell Signaling), and cleaved Caspase3 [5A1E] (Cell Signaling).

Techniques: Injection, Western Blot, Immunostaining

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

Article Title: Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

doi: 10.1073/pnas.1524575113

Figure Lengend Snippet: Neuronal Htt KO mice brain. (A) Photographs of H&E-stained coronal sections of the control and neuronal Htt KO mouse brain at the 90th day after tamoxifen injection. (Scale bar, 100 μm.) (B) Immunostaining of β-tubulin, cleaved caspase3, GFAP, and LC3I/II in the brains of neuronal Htt KO and control mice at the 90th day after tamoxifen induction. The relative numbers of NeuN- or GFAP-positive cells are shown below the images. TAM: tamoxifen. [Scale bar, 50 μm (Left), 200 μm (Right).] Ctl: heterozygous floxed Htt/Nestin-Cre mice injected with tamoxifen; oil: homozygous floxed Htt/Nestin-Cre mice injected with corn oil; KO: homozygous floxed Htt/Nestin-Cre mice injected with tamoxifen.

Article Snippet: Rabbit antibodies used in this study were obtained from commercial sources as follows: HTT [EPR5526] (Abcam), beta-tubulin III [EP1569Y] (Novus Biologicals), NF-κB [D14E12] (Cell Signaling), LC3I/II [NB100-2220] (Novus Biologicals), ELA3B [GTX105123] (GeneTex), FAT10 [EPR4370] (GeneTex), SPINK3 [2744S] (Cell Signaling), HA [C29F4] (Cell Signaling), p-NF-κB [93H1] (Cell Signaling), RIP3 [GTX107574] (GeneTex), Caspase3 [9662] (Cell Signaling), and cleaved Caspase3 [5A1E] (Cell Signaling).

Techniques: Staining, Injection, Immunostaining

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

Article Title: Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

doi: 10.1073/pnas.1524575113

Figure Lengend Snippet: Acute pancreatitis in 2-mo-old ubiquitous KO mice. (A) Photographs of abdominal cavity of 2-mo-old ubiquitous KO (CAG-CreER) and the heterozygous control mice after 3-d injections of tamoxifen. (Scale bar, 8 mm.) (B) Scheme outlining tamoxifen (TAM) injection followed by sacrificing (SAC) for analysis. (C) Western blots confirming Htt depletion in the pancreas tissues of ubiquitous KO mice. (D) Immunoblotting of pancreas lysates was performed with antibodies against cleaved caspase-3, P62, LC3I/II, CELA3B, and α-tubulin. The samples were obtained from ubiquitous KO and control mice (2 mo old) after i.p. tamoxifen injection for 3 d. (E) Serum amylase, serum lipase, pancreas trypsin activity, and myeloperoxidase (MPO) levels were measured 40 h after tamoxifen injection in ubiquitous KO and control mice. Data represent the means of eight control and ubiquitous KO mice at each interval ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001, four mice per group, n = 3 independent experiment, one-way ANOVA test). (F) Electron microscopy showing acinar cell degeneration in the pancreas of 2-mo-old ubiquitous KO mouse. (Scale bars, 2 μm.) Control is heterozygous floxed Htt/CAG-CreER mice injected with tamoxifen. Quantitative analysis of zymogen granules and degenerated cells is presented in Fig. S4D.

Article Snippet: Rabbit antibodies used in this study were obtained from commercial sources as follows: HTT [EPR5526] (Abcam), beta-tubulin III [EP1569Y] (Novus Biologicals), NF-κB [D14E12] (Cell Signaling), LC3I/II [NB100-2220] (Novus Biologicals), ELA3B [GTX105123] (GeneTex), FAT10 [EPR4370] (GeneTex), SPINK3 [2744S] (Cell Signaling), HA [C29F4] (Cell Signaling), p-NF-κB [93H1] (Cell Signaling), RIP3 [GTX107574] (GeneTex), Caspase3 [9662] (Cell Signaling), and cleaved Caspase3 [5A1E] (Cell Signaling).

Techniques: Injection, Western Blot, Activity Assay, Electron Microscopy

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

Article Title: Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

doi: 10.1073/pnas.1524575113

Figure Lengend Snippet: Interaction of Htt with Spink3 inhibits trypsin activity. (A) Western blotting (Upper) and quantification (Lower) of Spink3 in 2-, 4-, and 8-mo-old ubiquitous KO and control mice. (n = 3 independent experiments, two-way ANOVA test; n.s. represents no significant difference, ***P < 0.001). (B) Immunoprecipitation of Htt with Spink3 from 2-, 4-, and 8-mo-old WT mouse pancreas. More Spink3 in 2-mo-old than 4- or 8-mo-old mouse pancreas was precipitated with Htt. Anti–α-tubulin and IgG IP were used as control. n = 3 independent experiments. One-way ANOVA test, *P < 0.05; **P < 0.01; ***P < 0.001. (C) Schematic structures (Upper) of Spink3 and Htt proteins, and Western blots (Lower) showing coimmunoprecipitation of transfected GFP, GFP-N-Spink3, or GFP-C-Spink3 and truncated HTT (tHTT) in HEK293 cells. IgG immunoprecipitation served as controls. (D) Relative activities of trypsin in the control and ubiquitous KO mouse pancreatic acinar cells that were transfected with full-length Htt (fHTT), N-terminal Htt (nHTT), and truncated Htt lacking N-terminal 169 amino acids (tHTT). n = 3 independent experiments, two-way ANOVA test, P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit antibodies used in this study were obtained from commercial sources as follows: HTT [EPR5526] (Abcam), beta-tubulin III [EP1569Y] (Novus Biologicals), NF-κB [D14E12] (Cell Signaling), LC3I/II [NB100-2220] (Novus Biologicals), ELA3B [GTX105123] (GeneTex), FAT10 [EPR4370] (GeneTex), SPINK3 [2744S] (Cell Signaling), HA [C29F4] (Cell Signaling), p-NF-κB [93H1] (Cell Signaling), RIP3 [GTX107574] (GeneTex), Caspase3 [9662] (Cell Signaling), and cleaved Caspase3 [5A1E] (Cell Signaling).

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Transfection

Journal: PLoS ONE

Article Title: Bilateral Nerve Alterations in a Unilateral Experimental Neurotrophic Keratopathy Model: A Lateral Conjunctival Approach for Trigeminal Axotomy

doi: 10.1371/journal.pone.0070908

Figure Lengend Snippet: Representative histological photographs of corneal nerves stained with anti-βIII tubulin FITC-conjugated antibody. Normal subbasal (A) and stromal (E) nerve plexus. Subbasal nerve plexus completely disappeared (B; day 1, C; day 7, D; day 14) and stromal nerve definitely decreased from day1 (F; day 1, G; day 7, H; day 14) and after trigeminal axotomy. The nerve of the contralateral eye decreased from day 1 (J; day 1, K; day 7, L; day 14).

Article Snippet: Corneas were then stained with monoclonal NL637-conjugated anti-β-III tubulin antibody (anti-Neuron-specific β-III Tubulin-NL637, R&D systems Inc. Minneapolis, MN; dilution of 1∶100) at 4C degree overnight.

Techniques: Staining

Journal: PLoS ONE

Article Title: Bilateral Nerve Alterations in a Unilateral Experimental Neurotrophic Keratopathy Model: A Lateral Conjunctival Approach for Trigeminal Axotomy

doi: 10.1371/journal.pone.0070908

Figure Lengend Snippet: Representative histological photographs of corneal nerves stained with anti-βIII tubulin FITC-conjugated antibody. Normal subbasal (A) and stromal (E) nerve plexus.Subbasal nerve plexus completely disappeared (B; day 1, C; day 7, D; day 14) (A) and stromal nerve definitely decreased (B) from day 1 (F; day 1, G; day 7, H; day 14) after trigeminal axotomy. The nerve of the contralateral eye did not change (J; day 1, K; day 7, L; day 14).

Article Snippet: Corneas were then stained with monoclonal NL637-conjugated anti-β-III tubulin antibody (anti-Neuron-specific β-III Tubulin-NL637, R&D systems Inc. Minneapolis, MN; dilution of 1∶100) at 4C degree overnight.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Short Peptides Protect Fibroblast-Derived Induced Neurons from Age-Related Changes

doi: 10.3390/ijms252111363

Figure Lengend Snippet: List of primary antibodies for immunofluorescent staining live imaging analysis of functional activity of mitochondria and lysosomes.

Article Snippet: TUJ-1 , R&D Systems (Minneapolis, MN, USA) , MAB1195 , 1/2000.

Techniques: Staining, Imaging, Functional Assay, Activity Assay