Journal: Nucleic Acids Research

Article Title: The 26S proteasome drives trinucleotide repeat expansions

doi: 10.1093/nar/gkt295

Figure Lengend Snippet: Expansions are suppressed by siRNA knockdown of proteasome components in SVG-A cells. ( A and B ) Error bars represent ± SEM. (A) siRNA-mediated knockdown of proteasome subunits PSMC5 and PSMB3 but not DSS1 result in significant decreases in TNR expansion frequencies, * P < 0.05, compared with scrambled (Scr) control. For a summary of the data, see Supplementary Table S4 . A schematic of the 26S proteasome shows the location of these subunits. The second regulatory particle, at the ‘bottom’ of the core particle, was omitted for clarity. (B) Cell extracts were prepared after siRNA knockdown and assayed for chymotryptic activity of the proteasome as described in ‘Materials and Methods’ section. * P < 0.05, compared with scrambled control. Scr; n = 5, DSS1; n = 3, PSMB3; n = 4. ( C ) Representative immunoblot of polyubiquitinated proteins. Cells were treated with scrambled control siRNA (Scr) or siRNA to PSMB3, PSMC5 or DSS1, extracts were prepared and 10 µg total protein was analysed by immunoblot for polyubiquitinated proteins (Ubiquitin). Actin was used as a loading control. ( D ) Representative immunoblots for PSMC5 and PSMB3 knockdown. Fifty micrograms of total protein was loaded in each lane. Actin was used as a loading control. Additional knockdown data are presented in Supplementary Figure S6 . ( E ) Knockdown of DSS1 as measured by mRNA level, as described in ‘Materials and Methods’ section. Error bar denotes ± SEM, n = 3.

Article Snippet: Primary antibodies used were against ubiquitin (sc-8017, Santa Cruz), PSMC5 (NB100-345, Novus Biologicals), PSMB3 (PW8130, Biomol) and β-actin (A2066, Sigma).

Techniques: Knockdown, Control, Activity Assay, Western Blot, Ubiquitin Proteomics

Journal: Epigenetics & chromatin

Article Title: Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation.

doi: 10.1186/1756-8935-3-5

Figure Lengend Snippet: Figure 6 H3K27me3 is enhanced upon Sug1 knockdown. (a) Sug1 siRNA efficiently decreases endogenous Sug1. HeLa cells were transfected with control or Sug1-specific siRNA, harvested and subjected to western blot analysis for (top) endogenous Sug1 and (bottom) endogenous tubulin. (b-d) H3K27me3 is elevated at cytokine-inducible genes upon diminished Sug1 expression. ChIP assays were carried out in HeLa cells stimulated with IFN-g for 0 to 18 hours. Lysates were immunoprecipitated with control or endogenous H3K27me3 antibody. Associated DNA was isolated and analyzed as in Figure 2 using primers and probe spanning (b) CIITA pIV, (c) the MHC-II proximal promoter and (d) the GAPDH promoter. Data are presented as percentage input. IgG isotype control values were 0.003 ± 0.001 (CIITA pIV), 0.08 ± 0.02 (MHC-II promoter) and 0.01 ± 0.005 (GAPDH promoter). Values represent mean ± SEM of (n = 3) independent experiments.

Article Snippet: Sug1 antibody was from Novus Biologicals (Littleton, CO, USA).

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Immunoprecipitation, Isolation

Journal:

Article Title: PROTEASOME INHIBITION UP-REGULATES INFLAMMATORY GENE TRANSCRIPTION INDUCED BY AN ATYPICAL PATHWAY OF NF-?B ACTIVATION

doi: 10.1016/j.bcp.2009.10.006

Figure Lengend Snippet: (A). ILU-18 cells were either untreated or treated with 100μM PV for 4h, with or without pretreatment with 0.25μM Acla for 2 hours. ChIP assays employing anti-20S, anti-Sug1, and anti-RNA pol II were then performed using immunoprecipitated DNA amplified with primers specific for the IL-6 promoter. ChIP assay employing α-HA (irrelevant antibody) in cells treated with Acla+PV served as a specificity control.

Article Snippet: FK2 (Anti-Ubiquitin) and Sug1 and 20S proteasome antibodies were purchased from Biomol (Plymouth Meeting, PA).

Techniques: Immunoprecipitation, Amplification, Control