Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet: Cadmium indirectly inhibits the proteasome (A) Relative proteasome activity of HepG2 cells treated with CdCl 2 (Cd; + 1 μM or ++ 5 μM) or carfilzomib (CFZ, 10 nM) during 6 h. Each bar represents the mean, and error bars denote SD of at least three independent experiments. (B) Relative proteasome activity of purified 26S proteasome complexes incubated with the substrate Suc-LLVY-AMC and treated with or without Cd (1 μM) or CFZ (10 nM) during the indicated time in minutes (n = 3).

Article Snippet: Purified human proteasome complexes , Bio-Techne , E−365-025.

Techniques: Activity Assay, Purification, Incubation

Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet:

Article Snippet: Purified human proteasome complexes , Bio-Techne , E−365-025.

Techniques: Recombinant, Purification, Bicinchoninic Acid Protein Assay, Activity Assay, MTS Assay, Expressing, Western Blot, Plasmid Preparation, Software

Journal: Genes & Development

Article Title: A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation

doi: 10.1101/gad.305201.117

Figure Lengend Snippet: BRWD2/PHIP associates with CRL4, chromatin regulators, and active chromatin marks. ( A ) Silver-stained SDS-PAGE gel of Flag immunoprecipitations from control parental HEK293 TRex-expressing and Flag-BRWD2/PHIP-expressing cells. ( B ) Diagram summarizing BRWD2/PHIP protein–protein interactions. The solid black line indicates a strong interaction between BRWD2/PHIP and DDB1–CUL4A/B. Black dashed lines indicate interactions with chromatin remodelers and histones. Red dashed lines indicate interactions sensitive to treatment with MLN4924. ( C ) Western blot confirming BRWD2/PHIP interaction with CRL4 (DDB1 and CUL4A) and chromatin remodelers (CHD4 and PHF6). The middle band detected in the BRWD2/PHIP Western likely represents a BRWD2/PHIP protein cleavage product that occurs during immunoprecipitation incubation. ( D ) Western blot detecting histone modifications (H3K27ac, H3K4me1, H3K4me3, and H3K27me3) and histone variants (H2A.Z) associated with BRWD2/PHIP. Note that Flag-BRWD2/PHIP is expressed at near-endogenous levels and is not detectable with anti-Flag in the input but is detected after enrichment by immunoprecipitation.

Article Snippet: Other antibodies used in this study were H3K4me1 (Shilatifard Laboratory, no. 24), H3K4me2 (Shilatifard Laboratory, no. 27), H3K4me3 (Shilatifard Laboratory, no. 680), H3K27ac (Cell Signaling Technology, no. 8137), H2A.Z (Cell Signaling Technology, no. 2718), H3K27me3 (Shilatifard Laboratory, no. 67), DDB1 (Cell Signaling Technology, no. 6998), CUL4A (Cell Signaling Technology, no. 2699), CHD4 (Cell Signaling Technology #12011), PHF6 (Santa Cruz Biotechnology, no. sc-365237), β-Tubulin (E7, Developmental Studies Hybridoma Bank), Histone H3 (Shilatifard Laboratory, no. 42), and Flag-M2 (Sigma, no. F1804).

Techniques: Staining, SDS Page, Control, Expressing, Protein-Protein interactions, Western Blot, Immunoprecipitation, Incubation