Journal: Development (Cambridge, England)

Article Title: Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis

doi: 10.1242/dev.151555

Figure Lengend Snippet: AKT1 and AKT3 are phosphorylated in different types of spermatogonia. (A) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT1 Ser473 (P-AKT1) and GFRA1. GFRA+ cells in wild-type testes (arrows) and large clusters of GFRA+ cells in Tg(GDNF) testes (asterisks) are P-AKT1 negative. (B) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT3 Ser472 (P-AKT3) and GFRA1. Arrows indicate GFRA1+ P-AKT3+ As and Apr spermatogonia. Arrowheads indicate GFRA1+ P-AKT3+ cluster in Tg(Gdnf) tubules. See also Figs S5 and S6. Scale bars: 100 µm.

Article Snippet: Antibodies used were: mouse PLZF (Santa Cruz Biotech); rat GFRA1 (R&D Systems); rat LIN28A (a gift from Dr Eric G. Moss, UMDNJ, NJ, USA); rabbit SOHLH1 (Abcam); rabbit phospho-S6 (Ser240/244; #2215) and rabbit phospho-AKT1 (S473; D7F10) (Cell Signaling Technology); rabbit phospho-Akt3 (S472, #AP3468a) (Abgent); and mouse KIT (#CBL1360, Millipore) all at 1:200.

Techniques:

Journal: Development (Cambridge, England)

Article Title: Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis

doi: 10.1242/dev.151555

Figure Lengend Snippet: Effect of germ cell-specific deletion of Akt1 on germ cell development. (A-C) No significant change in body weight (A), testis weight (B) or sperm count (C) in 2-month-old mice when Akt1 is deleted in germ cells (n=3). (D) Paraffin wax-embedded sections of testis from control and mutant testis stained with PSA and probed with PLZF antibody. (E) Average PLZF+ cells per tubule (200-250 tubules counted) in control and mutant testis (n=3). (F) Akt1 transcript is significantly (***P=0.002) downregulated in mutant testis (n=3) as determined by qRT-PCR. (G) Absence of P-AKT1 staining in the mutant germ cells. Arrows indicate GFRA1+ P-AKT1+ (double-positive) cells. (H) Detection of P-AKT3 in Akt1-deleted germ cells. Arrows indicate GFRA1+ P-AKT3+ (double-positive) cells. Scale bars: 100 µm.

Article Snippet: Antibodies used were: mouse PLZF (Santa Cruz Biotech); rat GFRA1 (R&D Systems); rat LIN28A (a gift from Dr Eric G. Moss, UMDNJ, NJ, USA); rabbit SOHLH1 (Abcam); rabbit phospho-S6 (Ser240/244; #2215) and rabbit phospho-AKT1 (S473; D7F10) (Cell Signaling Technology); rabbit phospho-Akt3 (S472, #AP3468a) (Abgent); and mouse KIT (#CBL1360, Millipore) all at 1:200.

Techniques: Mutagenesis, Staining, Quantitative RT-PCR

Journal: Journal of Inflammation Research

Article Title: Role of Autophagy Induced by Pmel17 in the Pathogenesis of Vitiligo

doi: 10.2147/JIR.S551030

Figure Lengend Snippet: Down-regulation of Pmel17 inhibited the PI3K-AKT-mTOR signaling pathway. After transfection with Pmel17-siRNA for 48 hours, melanocytes were treated with the PI3K/AKT inhibitor LY294002 for 24 hours, ( a ) Detection of protein expression levels of p-AKT, p-mTOR, AKT and mTOR by Western blotting. ( b ) Detection of protein expression levels of LC3 and TYR by Western blotting. *P<0.05 indicates a significant difference compared with the control group.

Article Snippet: Rabbit anti-AKT (cat. no. 9272), rabbit anti-phosphorylated AKT (cat. no. 9271), rabbit anti-mTOR (cat. no. 2972), rabbit anti-phosphorylated mTOR (cat. no. 2971) and rabbit anti-P62 (cat. no. 5114) monoclonal antibodies were obtained from Cell Signaling Technology, Inc. Rabbit anti-LC3 antibody (cat. no. L7543) was obtained from Sigma.

Techniques: Transfection, Expressing, Western Blot, Control

Journal: Journal of Inflammation Research

Article Title: Role of Autophagy Induced by Pmel17 in the Pathogenesis of Vitiligo

doi: 10.2147/JIR.S551030

Figure Lengend Snippet: Diagram of melanin synthesis mechanism in Pmel17 knockdown melanocytes. After Pmel17 was knocked down, there were few fibrils in stage I melanosomes. In the stage II and stage III melanosomes, there was a disorder of the fibrillary matrix, and TYR cannot attach normally either. Meanwhile, the PI3K-AKT-mTOR pathway in the cytoplasm was inhibited, thereby activating autophagy and inducing the formation of autophagosomes. The melanosomes with structural abnormalities in stage II and stage III were phagocytosed and degraded by autophagosomes, eventually leading to a reduced melanin synthesis in melanocytes.

Article Snippet: Rabbit anti-AKT (cat. no. 9272), rabbit anti-phosphorylated AKT (cat. no. 9271), rabbit anti-mTOR (cat. no. 2972), rabbit anti-phosphorylated mTOR (cat. no. 2971) and rabbit anti-P62 (cat. no. 5114) monoclonal antibodies were obtained from Cell Signaling Technology, Inc. Rabbit anti-LC3 antibody (cat. no. L7543) was obtained from Sigma.

Techniques: Knockdown

Journal: Journal of Biological Chemistry

Article Title: Serine 129 Phosphorylation of α-Synuclein Induces Unfolded Protein Response-mediated Cell Death

doi: 10.1074/jbc.m802223200

Figure Lengend Snippet: FIGURE 2. Characterization of intracellular aggregations and their incidence in each cell line. A, the SH-SY5Y cells overexpressing WT -synuclein showed intracellular aggregations after 120 h of exposure to rotenone at 10 nM. The cells were co-stained with polyclonal phosphorylated -synuclein (Syn-p; panels a, d,andm),monoclonalphosphorylated-synuclein(Syn-m;panelsgandj),ERGIC(-COP;panelb,ERGIC-53;panele),ER(PDI;panelh),Golgi(NUCB;panelk),and lysosome (Lysotracker; panel n). In panels c, f, i, l, and o, the images were merged from each of left two ones, and the nuclei were stained with TO-PRO3 (blue). The scale bars indicate 10 m. B, cells were treated with the same concentration of rotenone for 24–120 h, and then the incidence of intracellular aggregations was quantified. For each sample, eight random fields were selected for counting. The results were analyzed by two-way analysis of variance test and were shown as the means S.E. *, p 0.05 versus both CAT and S129A; **, p 0.001 versus both CAT and S129A. C, sections from the locus ceruleus (panel a) and thedorsalvagalnucleus(panelb)frompatientswithPD.LBinthelocusceruleus(panela)andring-like(panelb,centerleft)andhomogenousLBs(panelb,center right) in the dorsal vagal nucleus were immunopositive for -COP. The scale bars indicate 10 m.

Article Snippet: Antibodies—The following primary antibodies were used in immunocytochemistry and Western blots: mouse monoclonal anti- -synuclein antibody (LB509; Zymed Laboratories Inc., South San Francisco, CA), rabbit polyclonal anti- -synuclein antibodies (AB5038; Chemicon, Temecula, CA), mouse anti- - synuclein phosphorylated at serine 129 monoclonal antibody (Pser#64; WAKO, Osaka, Japan), rabbit anti- -synuclein phosphorylated at serine 129 polyclonal antibodies (gift from Dr. Iwatsubo), mouse monoclonal anti- -COP antibody (Sigma), mouse monoclonal antiERGIC-53 antibody (LifeSpan Biosciences, Seattle, WA), mouse monoclonal anti- -tubulin antibody (Sigma), lysosome (Lysotracker; Invitrogen), rabbit polyclonal ER antibodies (PDI; Stressgen, Victoria, Canada), rabbit polyclonal Golgi antibodies (NUCB; AVIVA systems biology, San Diego, CA), rabbit polyclonal anti-activated caspase-9 antibodies (Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-activated caspase-3 antibodies (Cell Signaling Technology), rabbit polyclonal anti-phospho-IRE1 antibodies (Novus Biologicals, Littleton, CO), rabbit polyclonal anti-phosphoPERK antibodies (Santa Cruz Biotechnology), rabbit polyclonal antiphospho-eIF2 antibodies (Cell SignalingTechnology), rabbit polyclonal anti-c-JunN-terminal kinase (JNK) antibodies (Cell SignalingTechnology), and rabbit polyclonal anti-phospho-JNK antibodies (Cell Signaling Technology).

Techniques: Staining, Concentration Assay