Journal: PLoS ONE

Article Title: Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1α Protein under Hypoxic Conditions

doi: 10.1371/journal.pone.0033433

Figure Lengend Snippet: A. Hep3B cells were treated with 125 µM DMOG for 5 hours. Cytoplasmic and nuclear protein fractions were prepared. Twenty-five µg nuclear proteins and 50 µg cytoplasmic proteins were analyzed for SIRT1 and HIF-1α by Western blot. Sp1 was used as a nuclear fraction marker and α-tubulin for the cytoplasmic fraction. A representative blot of 3 independently performed experiments is shown. B. Cytoplasmic proteins (1250 µg) prepared in (A) were immunoprecipitated with a rabbit polyclonal SIRT1 antibody, chicken polyclonal HIF-1α antibody or a rabbit IgG1 control antibody. Immunoprecipitated proteins were subjected to Western blot analysis. A representative blot of 3 independently performed experiments is shown. C. SIRT1 inhibition leads to increased acetylation of HIF-1α. Hep3B cells were infected with lentiviral vectors containing shRNA against SIRT1 (shSIRT1_1958 or shSIRT1_3206) or scrambled shRNA (SHC002) as a negative control. Seven days post-infection, cells were incubated under hypoxia for 5 hours in the presence of 10 µM MG132. Whole cell lysates (2 mg) were immunoprecipitated with a mouse monoclonal HIF-1α antibody (2 µg) and precipitates were analyzed by Western blotting with a rabbit polyclonal anti-acetyl-lysine and a polyclonal chicken anti-HIF-1α antibody. Whole cell lysates (WCL: 100 µg) were used as input controls and were analyzed for SIRT1 and α-tubulin. A representative blot of 2 independently performed experiments is shown.

Article Snippet: Primary antibodies used were rabbit polyclonal anti-SIRT1 (MW: ∼120 kDa), mouse monoclonal anti-α-tubulin (MW: ∼55 kDa) and rabbit polyclonal anti-Sp1 (MW:∼100 kDa) all from Santa Cruz (Santa Cruz, USA), rabbit polyclonal anti-acetyl-lysine from Cell Signaling (Allschwil, Switzerland), mouse monoclonal anti-HIF-1α (MW:∼115 kDa) from Alexis Biochemicals (Lausen, Switzerland).

Techniques: Western Blot, Marker, Immunoprecipitation, Inhibition, Infection, shRNA, Negative Control, Incubation

Journal: eLife

Article Title: Vitamin D induces SIRT1 activation through K610 deacetylation in colon cancer

doi: 10.7554/eLife.86913

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Acetylated-lysine Rabbit Polyclonal , Cell Signaling , Cat# 9441 , 1:1000.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Mutagenesis, Protease Inhibitor, Software

Journal: BMC Biology

Article Title: 20-Hydroxyecdysone counteracts insulin to promote programmed cell death by modifying phosphoglycerate kinase 1

doi: 10.1186/s12915-023-01621-2

Figure Lengend Snippet: PGK1 knockdown at the wandering stage delays metamorphosis. A Morphology, HE staining, and caspase-3 location in the fat body after dsGFP and dsPGK1 knockdown. PGK1 knockdown (500 ng/larva administered in the sixth-instar at 72 h, three times over 24 h intervals). Images were obtained after the first RNAi exposure for 60 h. dsGFP was used as a negative control. Rabbit polyclonal antibodies against caspase-3 were used as the primary antibody. Green fluorescence indicates caspase-3 presence. Nuclei were stained with DAPI (blue). A’ Quantification of fluorescent caspase-3 in A . ** p < 0.01, n = 3. B TEM analysis of the fat body from A . LD indicates lipid droplets. Red arrows indicate autolysosomes or autophagosomes. Blue arrows represent the apoptotic nuclei. B’ Quantification of autophagosomes, autolysosomes, and apoptotic nucleus in B . * p < 0.05, n = 3. C and C’ The levels of ATG8-II and cleaved-caspase-3 in the fat body based on A . Western blot detection using anti-ATG8 and anti-caspase-3 antibodies. * p < 0.05, n = 3. D and D’ PGK1 acetylation levels in dsGFP and dsPGK1 fat body from A . PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with anti-PGK1 monoclonal antibody. * p < 0.05, n = 3. E Interference efficiency analysis of PGK1 at protein levels from the input of D . * p < 0.05, n = 3. F The Vmax of 3-PG production was determined from A in vitro. PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with the anti-PGK1 monoclonal antibody. n = 3. G and H Concentrations of glucose ( G ) and lactate ( H ) in H. armigera hemolymph from A . n = 3. I Phenotypes after PGK1 knockdown as A (500 ng/larva administered in sixth-instar at 72 h, three times over 24 h intervals). Images were obtained after the first RNAi exposure for 80 h. I’ Percentages of phenotypes in I based on three repeats (thirty larvae for each repeat). * p < 0.05, ** p < 0.01, n = 3. J The pupation time from 6th-6 h to pupa 0 d after RNAi exposure in I . * p < 0.05, n = 3. K The average body weight of pupa after RNAi exposure in I . n = 3. Bars indicate means ± SD for three independent experiments with five larvae per replicate. Statistical analyses were performed using two-tailed Student’s t tests (*: p < 0.05 and **: p < 0.01)

Article Snippet: Antibodies were commercially purchased including mouse monoclonal antibody anti-PGK1 (Santa Cruz Biotechnology, Cat. sc-130335; RRID: AB_627677), mouse monoclonal antibody anti-phosphate-tyrosine (anti-pY; Santa Cruz Biotechnology, Cat. sc-7020; RRID: AB_628123), rabbit polyclonal antibodies anti-acetylated lysine (anti-Ac; Cell Signaling Technology, Cat. 9441), rabbit monoclonal antibody anti-β-actin (ABclonal, Cat. AC026), mouse anti-GFP-Tag mAb (ABclonal, Cat. AE012), mouse anti-His-Tag mAb (ABclonal, Cat. AE003), mouse control IgG (ABclonal, Cat. AC011), and alkaline phosphatase-labeled goat anti-rabbit or horse anti-mouse IgG secondary antibodies (ZSGB-BIO, Cat. ZB-2308; ZB-2310).

Techniques: Staining, Negative Control, Fluorescence, Western Blot, Purification, In Vitro, Two Tailed Test

Journal: Cell reports

Article Title: Downregulation of hepatic ceruloplasmin ameliorates NAFLD via SCO1-AMPK-LKB1 complex

doi: 10.1016/j.celrep.2022.111498

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-acetylated-Lysine antibody, WB, dil: 1/1,000 , Cell Signaling Technology , Cat#9441; RRID: AB_331805.

Techniques: Magnetic Beads, shRNA, Recombinant, Protease Inhibitor, Lysis, Modification, Transfection, BIA-KA, Mutagenesis, Purification, Reporter Assay, TUNEL Assay, RNA Sequencing Assay, Transgenic Assay, Plasmid Preparation, Software