Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: ( A ) In vivo acetylation assay in HEK293T cells transfected with plasmids coding for Myc tagged Gli2 and the indicated HATs. Cell extracts were immunoprecipitated with anti-Myc antibody and acetylation was revealed with anti-acetyl lysine antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. Empty, pcDNA3. ( B ) Luciferase assay in HEK293T. Cells were transfected with 12× Gli-Luc and TK renilla reporter vectors, Gli2, p300, TIP60, pCAF where indicated. *p<0.01 vs Empty (pcDNA3). Results are shown as the average ± SD of triplicate experiments (n = 4). ( C ) (Top) ClustalW alignment of the region surrounding lysine 757 of Gli2 in different species. Identical, strongly conservative and weakly conservative aminoacids are indicated by asterisk, colon and dots respectively, according to ClustalW convention. (Bottom) In vivo acetylation assay in HEK293T cells transfected with Myc-Gli2 WT or K757R and p300 where indicated. Acetylation was detected as previously described. ( D ) (Top) Luciferase assay performed in HEK293T cells transfected with 12× Gli-Luc, TK renilla, Myc-Gli2 WT or the acetylation mimetic mutant Myc-Gli2 K757Q. *p<0.01 vs Empty; **p<0.05 vs WT. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Western blot analysis of Myc-Gli2 WT and K757Q expression levels.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: In Vivo, Acetylation Assay, Transfection, Immunoprecipitation, Western Blot, Luciferase, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: ( A ) K757 acetylation of wild type Myc tagged Gli2 and K757R mutant in HEK293T cells. Cells extract were immunoprecipitated with anti-Myc antibody and acetylation was detected by anti acetyl-Gli2(Lys757) specific antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. ( B ) Acetylation of endogenous Gli2 in NIH3T3 cells, treated with SAG or DMSO for 24 hours. Acetylation was detected with anti acetyl-Gli2(Lys757) specific antisera. ( C ) (Top) Luciferase assay in NIH3T3 cells transfected with 8× Gli-Luc reporter, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Gli1 mRNA levels normalized with the housekeeping HPRT mRNA in NIH3T3 cells transfected with Myc-Gli2 WT and K757R mutant. ( D ) Transcriptional activity of Myc-Gli2 WT and K757R mutant in response to SAG treatment: Ptch1 mRNA levels (QPCR), normalized with the housekeeping GAPDH mRNA in 24 hours SAG-treated NIH3T3 cells, transfected with Empty vector, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty, **p<0.05 vs DMSO; Results are shown as the average ± SD of triplicate experiments (n = 3).

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Luciferase, Transfection, Activity Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: Immunohistochemical staining of Gli1(upper-right), acetyl-Gli1(Lys518) (bottom-left), acetyl-GLi2(Lys757) (bottom-right), or Hematoxylin (upper-left) in a 6-day-old mouse cerebellum.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: ( A ) Cellular fractionation of NIH3T3 cells shows no difference in the localization of Myc tagged Gli2 wild type (WT), K575R and K575Q mutants. Purity of fractionation documented by tubulin (cytoplasmic, Cyto) and CREB (nuclear, Nu) staining. ( B ) Promoter occupancy of Gli2 is prevented by K757 acetylation. HEK293T cells were transfected with Myc tagged Gli2 wild type (WT), K757R, K757Q and Empty vectors and Chromatin Immunoprecipitation (ChIP) was carried out. Quantitative real-time PCR was performed using primers encompassing the Gli-BS of human Ptch1 promoter (bottom, schematic representation). Results are indicated as fold difference, relative to Empty (pcDNA3) control. *p<0.05 WT vs Empty, **p<0.05 K757Q vs WT. Results are shown as the average ± SD of triplicate experiments (n = 4). Western blot analysis with anti Myc antibody showing equivalent amounts of Myc-Gli2 WT and mutants. Empty, pcDNA3.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: Cell Fractionation, Fractionation, Staining, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: Following Hh/Ptch interaction, Smo triggers a signaling cascade leading to Gli2 deacetylation and to the inhibition of the βTrCP-regulated balance between Gli2R and full length Gli2 (Gli2A). Both events contributes to the early signal-dependent activation of the Hh pathway. Once activated, Gli2 promotes transcription of Gli1, whose activity is also regulated by Hh-induced HDAC1-mediated deacetylation, thus generating a positive feedback loop (late activation).

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: Inhibition, Activation Assay, Activity Assay