Journal: Redox Biology

Article Title: Targeting GPX4-mediated ferroptosis protection sensitizes BRCA1 -deficient cancer cells to PARP inhibitors

doi: 10.1016/j.redox.2024.103350

Figure Lengend Snippet: BRCA1/BARD1 catalyzes K6-linked polyubiquitination of GPX4 on lysine 47 and 58. (A) Flag-GPX4 was co-transfected with or without HA-ubiquitin (Ub) into UWB1.289/UWB-BRCA1 cells, and then treated with 5 μM MG132 for 6 h before harvesting for analysis of flag-GPX4 ubiquitination. (B) A2780 cells were co-transfected with indicated plasmids, IP was performed using anti-flag affinity resin after MG132 treatment for 6 h and ubiquitination of flag-GPX4 was analyzed by Western blot. (C) A2780 cells with or without BRCA1 knockdown were co-transfected with indicated plasmids, ubiquitination of flag-GPX4 was analyzed by IP with anti-flag affinity resin after MG132 treatment. (D–E) Purified His-GPX4 was incubated in presence of Ub/E1/E2, purified flag-BARD1, and either WT or I26A mutant flag-BRCA1 1-303 protein, then his-GPX4 ubiquitination was analyzed. (F–G) WT, K11R, K47R or K58R flag-GPX4 was co-transfected with WT (F) or K6-specfic(G) HA-ubiquitin, along with or without myc-BRCA1/BARD1 into A2780 cells, then flag-GPX4 ubiquitination was analyzed after MG132 treatment. (H) WT or K47RK58R (2 KR) flag-GPX4 was co-transfected with HA-Ub and myc-BRCA1/BARD1 complex, followed by analysis of flag-GPX4 ubiquitination after MG132 treatment.

Article Snippet: Ubiquitin Activating Enzyme E1/UBA1 protein (11990-H20B-50) was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Transfection, Western Blot, Knockdown, Purification, Incubation, Mutagenesis

Journal: Nature Structural & Molecular Biology

Article Title: Structure and activation of the RING E3 ubiquitin ligase TRIM72 on the membrane

doi: 10.1038/s41594-023-01111-7

Figure Lengend Snippet: a , Ubiquitination activity of TRIM72 in the presence or absence of PS liposomes. b , Increased ubiquitination activity of TRIM72 Q57R in the presence or absence of PS liposomes. c , Ubiquitination activity of TRIM72 Q57R and TRIM72 Q57R/L74R in the presence or absence of PS liposomes. d , Ubiquitination activity of endogenous microvesicle-bound or free-solution states of TRIM72 Q57R and TRIM72 Q57R/L74R . e , Ubiquitination activity of RING and 2×RING constructs with TRIM72 WT and TRIM72 Q57R . f , Ubiquitination activity of RING and 2×RING constructs with TRIM72 Q57R and TRIM5α. TRIM72 proteoliposomes were reconstituted and further separated by SEC ( a – c ). TRIM72 microvesicles were purified from HEK293T cells and isolated by SEC ( d ). Poly-Ub is represented as a polyubiquitination ladder. Bands close to 140 kDa were assumed to be ubiquitin-like modifier activating enzyme 1 (UBA1)~Ub. Ub was detected with an anti-Ub antibody. Strep-tagged TRIM72 or intact TRIM72 was detected using an anti-Strep-tag antibody or an anti-TRIM72 antibody, respectively ( a – d ). The superfolder variant of green fluorescent protein (sfGFP)-fused RING and 2×RING constructs were detected with an anti-GFP antibody ( e , f ). Phospholipids were stained with Sudan Black B ( a – c ). Microvesicles were detected with an anti-Na + –K + ATPase α1 antibody ( d ). Independent experiments were performed in triplicate. TRIM72 WT, green; TRIM72 Q57R , pink; TRIM72 Q57R/L74R , cyan; TRIM72 RING_WT, yellow; TRIM72 RING_Q57R, light pink; TRIM5α RING, magenta.

Article Snippet: Plasmids encoding UBA1 (human UBA1, Addgene, 34965) and UBE2D3 (human UBE2D3, Addgene, 15784) were obtained from Addgene ( http://addgene.org ).

Techniques: Ubiquitin Proteomics, Activity Assay, Liposomes, Construct, Purification, Isolation, Strep-tag, Variant Assay, Staining

Journal: Proteome Science

Article Title: A proteomic analysis of C-reactive protein stimulated THP-1 monocytes

doi: 10.1186/1477-5956-9-1

Figure Lengend Snippet: Differentially expressed proteins in LPS-, mCRP- or pCRP-treated THP-1 monocytes.

Article Snippet: Polyclonal antibodies against HSP105 was obtained from MBL (Woburn, MA, USA), UBE1 from Atlas Antibodies (Stockholm, Sweden) and Hsp70(Hsp72) from Stressgen (MI, USA).

Techniques: Ubiquitin Proteomics

Journal: Proteome Science

Article Title: A proteomic analysis of C-reactive protein stimulated THP-1 monocytes

doi: 10.1186/1477-5956-9-1

Figure Lengend Snippet: Selected differentially expressed proteins are tabulated with mean values for three independent analyses and standard deviation of spot density.

Article Snippet: Polyclonal antibodies against HSP105 was obtained from MBL (Woburn, MA, USA), UBE1 from Atlas Antibodies (Stockholm, Sweden) and Hsp70(Hsp72) from Stressgen (MI, USA).

Techniques: Standard Deviation, Control

Journal: Proteome Science

Article Title: A proteomic analysis of C-reactive protein stimulated THP-1 monocytes

doi: 10.1186/1477-5956-9-1

Figure Lengend Snippet: Quantitative analysis of differentially expressed proteins . A. mCRP induces expression of cellular stress related proteins of the ubiquitin/proteasome system and the heat shock response. Expression levels of several proteins of interest were quantified and are given in the graphs as mean +/- standard deviation. # indicates p < 0.05 compared to control. mCRP up-regulates UBE1, and HSP 70, as well as the alpha isoform of HSP 105. pCRP induces the beta isoform of HSP105. B. Differential expression of proteins involved in the inflammatory response. ACTN4 and RIPK2 are increased in the LPS, as well as in the m- and pCRP group. ENOA is increased by LPS and mCRP stimulation of THP-1 monocytes, whereas EHD1 is significantly upregulated after stimulation with LPS. C. Western blot analysis of selected proteins that are differentially expressed in THP-1 cells. Selected Proteins were validated by Western blotting. Western blot results are shown exhibiting specific staining of 1D-gels with anti-HSP70, anti-RIPK2 and anti-UBE1 antibodies. Analysis served as a confirmation of the protein identity in addition to the Mass-spec analysis.

Article Snippet: Polyclonal antibodies against HSP105 was obtained from MBL (Woburn, MA, USA), UBE1 from Atlas Antibodies (Stockholm, Sweden) and Hsp70(Hsp72) from Stressgen (MI, USA).

Techniques: Expressing, Ubiquitin Proteomics, Standard Deviation, Control, Quantitative Proteomics, Western Blot, Staining, Mass Spectrometry