Journal: Redox Biology

Article Title: ROS disrupt COP9 signalosome-mediated ABCA1 protection and trigger its ubiquitination and degradation by cullin3 inhibiting cholesterol efflux and promoting foam cell formation in response to GPCR agonists

doi: 10.1016/j.redox.2026.104095

Figure Lengend Snippet: Forced expression of CSN5 restores ABCA1 levels and cholesterol efflux from thrombin-induced downregulation and diminishes foam cell formation. A and B, RAW264.7 cells (A) and MASMCs (B) were transfected with pCMV or pCMV-CSN5 plasmid, treated with and without thrombin (0.5 U/ml) for 4 h, cell extracts were prepared, immunoprecipitated with anti-ABCA1 antibodies and the immunocomplexes were immunoblotted for the indicated proteins using their specific antibodies. The input protein was immunoblotted for CSN1, CSN2, CSN5, Myc-tag and β-actin. C–F, All the conditions were the same as in panel A and B except that cells were assayed for cholesterol efflux (C and D) or foam cell formation (E and F). The bar graphs represent Mean ± SD values of three biologically independent experiments. ∗p < 0.01 versus pCMV + vehicle; ∗∗p < 0.01 versus pCMV + thrombin. Scale bar in panels E and F is 20 μm.

Article Snippet: MycDDK-tagged pCMV-CSN5 plasmid (MR204941) was bought from Origene (Rockville, MD).

Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Redox Biology

Article Title: ROS disrupt COP9 signalosome-mediated ABCA1 protection and trigger its ubiquitination and degradation by cullin3 inhibiting cholesterol efflux and promoting foam cell formation in response to GPCR agonists

doi: 10.1016/j.redox.2026.104095

Figure Lengend Snippet: Ang II inhibits cholesterol efflux and increases foam cell formation by downregulation of ABCA1 levels. A, Quiescent RAW264.7 cells were treated with and without Ang II (1 μM) for the indicated time periods, cell extracts were prepared and analyzed by Western blotting for ABCA1, ABCG1, SR-B1, CSN5, cullin3, and β-actin levels using their specific antibodies and quantified. B, Quiescent RAW264.7 cells were treated with and without Ang II (1 μM) in the presence or absence of allopurinol (50 μM) for indicated time-periods, cell extracts prepared, immunoprecipitated with anti-ABCA1 antibody and the immunocomplexes were analyzed by Western blotting for the indicated proteins using their specific antibodies. The input protein was immunoblotted for β-actin. C, Quiescent RAW264.7 cells were treated with and without Ang II in the presence or absence of allopurinol for 4 h, cell extracts prepared, immunoprecipitated with anti-ABCA1 antibody and the immunocomplexes were analyzed by Western blotting for the indicated proteins using their specific antibodies. The input protein was immunoblotted for β-actin. D, All the conditions were same as in panel C except that the cells were subjected to in situ PLA. E, Quiescent RAW264.7 cells were treated with and without Ang II (1 μM) in the presence or absence of allopurinol (50 μM) for 4 h and assayed for H 2 O 2 production using Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit. F, All the conditions were same as in panel A except that cell extracts were analyzed by Western blotting for xanthine oxidase, and β-actin levels using their specific antibodies and quantified. G and H, All the conditions were the same as in panel E except that the cells were assayed for cholesterol efflux (G) or foam cell formation (H). I, RAW264.7 cells were transfected with pCMV or pCMV-CSN5 plasmid, treated with and without Ang II (1 μM) for 4 h, cell extracts were prepared, immunoprecipitated with anti-ABCA1 antibodies and the immunocomplexes were immunoblotted for the indicated proteins using their specific antibodies. The input protein was immunoblotted for CSN1, CSN2, CSN5, Myc-tag and β-actin. J and K, All the conditions were the same as in panel I except that cells were assayed for cholesterol efflux (J) or foam cell formation (K). The bar graphs represent Mean ± SD values of three biologically independent experiments. ∗p < 0.01 versus control; ∗∗p < 0.01 versus Ang II. Scale bar in panel D is 10 μm and panels H and K is 20 μm.

Article Snippet: MycDDK-tagged pCMV-CSN5 plasmid (MR204941) was bought from Origene (Rockville, MD).

Techniques: Western Blot, Immunoprecipitation, In Situ, Transfection, Plasmid Preparation, Control

Journal: iScience

Article Title: Alox15 via H 2 O 2 mediates TP receptor palmitoylation and its membrane trafficking leading to platelet activation

doi: 10.1016/j.isci.2026.114796

Figure Lengend Snippet: 12/15-LOX via COP9 signalosome protects XO from proteasomal degradation (A and B) Platelets from WT and 12/15-LOX −/− mice were treated with and without F 2 -TXA2 for 30 min, and mRNA or protein extracts were prepared and analyzed by qRT-PCR (A) or western blotting (B) for XO and β-actin mRNA or protein levels, respectively, using their specific primers or antibodies ( n = 3). (C) Platelets from WT mice were incubated with and without F 2 -TXA2 in the presence and absence of rapamycin (100 nM) for 30 min, and protein extracts were analyzed by western blotting for XO and β-actin levels using their specific antibodies ( n = 3). (D) All the conditions were same as in (B) except that the protein extracts were immunoprecipitated with anti-XO antibody and the immunocomplexes were analyzed by western blotting using anti-ubiquitin antibody. The blot was sequentially reprobed for CSN5, cullin 2, and XO. The input protein was analyzed by western blotting for CSN5, cullin 2, and β-actin levels. All data are presented as mean ± SD and analyzed by paired Student’s t test. ∗ p < 0.01 versus control; # p < 0.01 versus F 2 -TXA2.

Article Snippet: Anti-CSN5 antibody , Cell Signaling Technology , 6895.

Techniques: Quantitative RT-PCR, Western Blot, Incubation, Immunoprecipitation, Ubiquitin Proteomics, Control

Journal: iScience

Article Title: Alox15 via H 2 O 2 mediates TP receptor palmitoylation and its membrane trafficking leading to platelet activation

doi: 10.1016/j.isci.2026.114796

Figure Lengend Snippet: ROS plays a role in F 2 -TXA2-induced platelet activation in humans (A) Human platelets were treated with and without F 2 -TXA2 in the presence and absence of ML351 for 30 min and protein extracts were prepared and analyzed by western blotting for XO and β-actin levels using their specific antibodies ( n = 3). (B) Human platelets were treated with and without F 2 -TXA2 in the presence and absence of ML351 for 30 min and H 2 O 2 production was measured ( n = 4). (C) All the conditions were same as in (A) except that the protein extracts were immunoprecipitated with anti-XO antibody and the immunocomplexes were analyzed by western blotting using anti-ubiquitin antibody. The blot was sequentially reprobed for CSN5, cullin 2, and XO. The input protein was analyzed by western blotting for CSN5, cullin 2, and β-actin levels. (D) Human platelets were treated with and without F 2 -TXA2 in the presence and absence of allopurinol (50 μM) for 30 min and H 2 O 2 production was measured ( n = 4). (E) Human platelets were treated with and without F 2 -TXA2 in the presence and absence of allopurinol for 30 min and subjected to Acyl-RAC assay as described in B. (F) Human platelets were treated with and without F 2 -TXA2 in the presence and absence of allopurinol for 30 min, labeled with sulfo-NHS-SS-Biotin and membrane and soluble fractions were isolated and analyzed by western blotting for TP receptor, Na/K ATPase, and MEK1 levels using their specific antibodies. (G and H) All the conditions were the same as in (D) except that the platelets were subjected to adhesion (G) and the PRP was assayed for platelet aggregation (H) ( n = 3). (I and J) Human platelets (I) or PRP (J) were incubated with and without H 2 O 2 (200 μM) or uric acid (100 μM) for 30 min, and the platelets were subjected to adhesion (I) and the PRP was assayed for platelet aggregation (J) ( n = 3). All data are presented as mean ± SD and analyzed by paired Student’s t test. ∗ p < 0.01 versus control; # p < 0.01 versus F 2 -TXA2 or H 2 O 2 .

Article Snippet: Anti-CSN5 antibody , Cell Signaling Technology , 6895.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Labeling, Membrane, Isolation, Incubation, Control