Journal: The Journal of Cell Biology

Article Title: Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins

doi: 10.1083/jcb.200803022

Figure Lengend Snippet: Although long-lived proteins are not degraded more rapidly after exposure to paraquat, they are oxidatively damaged, and a lack of SOD1 also enhances the degradation of recently synthesized proteins. (A) Yeast were grown exponentially, and a portion of cells were treated with cycloheximide for 2 h to allow the degradation of short-lived proteins (the times studied in ). Both the control and treated cells were then exposed to paraquat for 90 or 150 min. The presence of carbonylated proteins in equal amounts of cell proteins was assayed after derivitization with DNP-hydrazine (DNPH) and then Western blotting with an anti–DNP-hydrazone antibody. A control lane without the treatments with DNPH and paraquat is included to show the specificity of the antibody. Equal loading of lanes was shown with an eIF5A antibody. (B) WT, Δsod1 mutant, and the Δsod1 mutant expressing SOD1 from a plasmid were labeled with 35 S-Met for 5 min at 30°C. Rates of protein degradation were measured at 30°C as in .

Article Snippet: Centromeric plasmid expressing SOD1 (pLS101; ) was provided by V. Culotta (Johns Hopkins School of Public Health, Baltimore, MD).

Techniques: Synthesized, Control, Western Blot, Mutagenesis, Expressing, Plasmid Preparation, Labeling

Journal: PLoS ONE

Article Title: PPARγ Regulates Expression of Carbohydrate Sulfotransferase 11 ( CHST11/C4ST1 ), a Regulator of LPL Cell Surface Binding

doi: 10.1371/journal.pone.0064284

Figure Lengend Snippet: (A) Chip-Seq data of PPARγ and RXRα occupancy on the Chst11 gene according to Nielssen et al. . UCSC Genome Browser tracks at day 0, 1, 2 4 and 6 of differentiation are shown. Please note differences in y-axes. Two intronic PPARy-RXRα binding sites were designated site 1 and 2. (B) ChIP-PCR on 3T3-L1 preadipocytes and adipocytes. Chromatin was prepared on day 0 and day 6 of differentiation and subjected to immunoprecipitation using antibodies against PPARγ. Enriched DNA was analysed using quantitative PCR with primers located at site 1 and 2 in the Chst11 gene (dark gray and blacks bars, respectively). As a positive control, primers located at the Fabp4 PPREs (∼5500 bp from transcription start site; light gray bars) were used, primers located in the globin locus were used as a negative control (white bars). Results are shown relative to normalized ChIP recovery data of day 0 and results are representative of at least 3 independent experiments. (C and D) U2OS cells were cotransfected with a reporter construct (TKpGL3) containing Chst11 site 1 or site 2 sequences, or the parental reporter construct, together with empty (pCDNA) or PPARγ encoding expression vectors. Activation of the luciferase reporter in the absence or presence of 1 µM rosiglitazone is expressed as fold induction over that with empty reporter cotransfected with pCDNA in the absence of rosiglitazone after normalisation for Renilla luciferase activity. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (E) Chst11 mRNA expression in 3T3-L1 adipocytes that had been treated with control or PPARy siRNA from the start of differentiation and analyzed at day 5. Relative mRNA expression levels were related to control siRNA treated cells and normalized for the TFIIb reference gene. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (F) Chst11 mRNA expression in 3T3-L1 adipocytes and the effect of rosiglitazone treatment (1 µM, 24 h). Relative mRNA expression levels were normalized for the TFIIb reference gene. (G) As in panel F, but after treatment with the PPARγ antagonist GW9662 (10 µM, 24 h).

Article Snippet: In short, cells were transfected in 24-well plates with 1 µg reporter plasmid, 10 ng PPARy expression construct, and 2 ng pCMV-Renilla reporter plasmid (Promega).

Techniques: ChIP-sequencing, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Construct, Expressing, Activation Assay, Luciferase, Activity Assay