rbx2 flox allele  (Jackson Laboratory)

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Schematic of APEX2-catalyzed biotinylation at the mitochondrial outer membrane (MOM) via fusion to a mitochondrial targeted peptide derived from MAVS (mitochondrial antiviral-signaling protein). Overlap of APEX2-MOM data with the ESI (E3-substrate interaction) network reveals the association of CRLs with mitochondria. B , Representative Western blot of biotinylated proteins in neonatal rat ventricular cardiomyocytes (NRVCs) with adenoviral (Ad) expression of APEX2-MOM. NRVCs were treated with CCCP (10 µM) before H 2 O 2 activation. The resultant biotinylated proteins were enriched by streptavidin beads. C , Representative Western blot of cytosol and mitochondrial fractions from NRVCs treated with CCCP (10 µM) for the indicated times. Arrowhead, neddylated CUL5. Tubulin and VDAC serve as cytosol and mitochondrial markers, respectively. D , Immuno-gold electron microscopic images showing the localization of RBX2 on mitochondrial membranes (arrowheads) in NRVCs expressing HA-RBX2. NRVCs infected with Ad-GFP and Ad-HA-OMP25 (MOM protein) serve as negative and positive controls, respectively. Scale bars, 0.5 µm. E , Representative Western blot of mitochondria with or without proteinase K (PK) treatment for 30 min on ice after isolation from NRVCs. F , Confocal images showing the colocalization (arrowhead) of RBX2 with mitochondria in CCCP-treated cardiomyocytes. NRVCs with adenoviral expression of HA-RBX2 were treated with CCCP for 3 hours and stained or immunostained as indicated. HA, green. Mitotracker, red. TOMM20, blue. Line scan co-localization analysis was done for all channels. Scale bars, 50 µm.

Article Snippet: Cardiomyocyte-specific RBX2 knockout (RBX2 CKO ) mice were generated by crossing a Rbx2 Flox allele with Exon 1 flanked with loxP sites with αMHC Cre/+ mice (the Jackson Laboratory, strain #011038).

Techniques: Membrane, Derivative Assay, Western Blot, Expressing, Activation Assay, Infection, Isolation, Staining

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Schematics of creation of tamoxifen-inducible, cardiac-specific RBX2 knockout (iCKO) mice. B , Western blot of indicated proteins in mouse hearts at 12 days after tamoxifen injection. C , Quantification of B . D , Survival curve. E , Gross morphology of mouse heart (top) and hematoxylin and eosin staining of myocardium section (bottom) at 12 days after tamoxifen injection. F , Heart weight to tibial length ratio and lung weight to tibial length ratio. F/F: n=13, MCM: n=5, iCKO: n=18. G , Representative B-mode images. H , Quantification of echocardiographic parameters before (F/F: n=17, MCM: n=6, iCKO: n=23) and after (F/F: n=12, MCM: n=6, iCKO: n=12) tamoxifen treatment. I , Wheat germ agglutinin (WGA) staining (left) of myocardium sections and quantification of cardiomyocyte (CM) cross-sectional area (right). More than 100 cells/heart and 3 and 12 hearts from F/F and iCKO mice, respectively, were quantified. J , Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of myocardium sections (left) and quantification (right). Three fields per heart, and 3 hearts per group, were quantified. K , qPCR analysis of the indicated genes. F/F: n=4, iCKO: n=4. One-way ANOVA followed by post hoc Tukey test was used in C , F and H . Log-rank (Mantel-Cox) test in D . Nested t test in I and J . Student t test in K . * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Cardiomyocyte-specific RBX2 knockout (RBX2 CKO ) mice were generated by crossing a Rbx2 Flox allele with Exon 1 flanked with loxP sites with αMHC Cre/+ mice (the Jackson Laboratory, strain #011038).

Techniques: Knock-Out, Western Blot, Injection, Staining, TUNEL Assay

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Scheme of procedures for identification Rbx2-regulated proteome in NRVCs. NRVCs were transfected with indicated siRNAs, followed by CCCP (10 µM) treatment for 6 hours. Cell lysates were collected for trypsin digestion. The resultant peptides were labeled by TMT before mass spectrometry analysis. B , Principal component analysis (PCA) of normalized protein expression in whole cell proteome. CTL, siLuci. KD, siRBX2. C , Analysis of the percentage of differential expression of proteins (DEPs) in each group. D, Volcano plot of differentially expressed proteins (Sig., blue and red) in CCCP-treated RBX2-deficient CMs (KD) compared with CCCP-treated CTL. E , Venn diagram showing overlap of RBX2-regulated proteome and mitochondrial proteins annotated in MitoCarta 3.0. F , Venn diagram showing the identification of RBX2-regulated MOMs. G , Heatmap showing the relative expression of MOM proteins amongst the groups of cells. H , Western blot of MOM proteins in control and RBX2-deficient cardiomyocytes. N=4 for each group. Multiple t test was used. ** P <0.01, *** P <0.001, and **** P <0.0001. I , Western blot of WT and RBX2KO Hela cells treated with cycloheximide (CHX, 100 nM) for indicated times. RBX2 was deleted in Hela cells via CRISPR/Cas9-mediated gene editing. J , WT and RBX2KO Hela cells were transfected with plasmids expressing HA-Ub (pCDNA3-HA-Ub), treated with a proteasome inhibitor Bortezomib (BZM, 100 nM) for 6 hours, and subjected to immunoprecipitation followed by Western blot.

Article Snippet: Cardiomyocyte-specific RBX2 knockout (RBX2 CKO ) mice were generated by crossing a Rbx2 Flox allele with Exon 1 flanked with loxP sites with αMHC Cre/+ mice (the Jackson Laboratory, strain #011038).

Techniques: Transfection, Labeling, Mass Spectrometry, Expressing, Western Blot, CRISPR, Immunoprecipitation

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: Adult MCM and RBX2 iCKO (iCKO) mice were administered with tamoxifen (50 mg/kg/d for 5 days). Tissues were collected for indicated analyses ( A - H ) at 12 days after tamoxifen injections. A , Representative confocal images (left) of MCM and RBX2 iCKO myocardium sections immunostained with pUb (green), HSP60 (red, mitochondrial marker) and DAPI. Scale bars, 10 µm. B , Quantification of pUb+ foci normalized by mitochondria (HSP60+) area. A total of 16 views from two hearts per group were quantified. C - D , Western blot ( C ) and quantification ( D ) of mitochondrial (mito) and cytosolic (cyto) P62 in mouse hearts. E - F , Western blot ( E ) and quantification ( F ) of mitochondrial (mito) and cytosolic (cyto) LC3-II in mouse hearts. Mice at 12 days after tamoxifen administration were intraperitoneally injected with bafilomyocin A1 (BFA, 3 µmol/kg) for 3 hours before tissue harvest. G , Representative confocal images of mt-Keima at 488 nm and 568 nm, respectively, in epicardial cardiomyocytes and the derived heatmaps. Neonatal MCM and RBX2 iCKO mice were transduced with AAV9-mt-Keima (1X10 GC/pup). At 10 weeks of age, mice were treated with tamoxifen and intact mouse hearts were excised 12 days later and scanned for mt-Keima signals in epicardial cardiomyocytes in situ with confocal microscope. Scale bars, 20 µm. H , Quantification of relative 568/488 ratio. 20-40 views per heart, 3 hearts per group were quantified. I , Western blot of indicated proteins in adult cardiomyocytes isolated from 2-month-old CTL (RBX2 F/F ) or RBX2 CKO (CKO) mouse hearts. Results from two different batches of cells are shown. Nested t test was was used in B and H , Mann-Whitney test in D , and One-way ANOVA in F. * P < 0.05, ** P < 0.01, **** P <0.0001.

Article Snippet: Cardiomyocyte-specific RBX2 knockout (RBX2 CKO ) mice were generated by crossing a Rbx2 Flox allele with Exon 1 flanked with loxP sites with αMHC Cre/+ mice (the Jackson Laboratory, strain #011038).

Techniques: Marker, Western Blot, Injection, Derivative Assay, Transduction, In Situ, Microscopy, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Western blot of Parkin in NRVCs. Cells were infected with Ad-Parkin and transfected with indicated siRNAs. B , Western blots (left) and quantification (right) of pS65-Ub. Neonatal mouse ventricular CMs (NMVCs) were isolated from WT or Parkin -/- mouse hearts, transfected with siRNA, and treated with CCCP (10 µM) for 12 hours. B , Quantification of phosphorylated Ub. C , Western blots of cell lysates from NRVCs transfected with siRNAs and treated with CCCP (10 µM). D , Western blots of cell lysates from NRVCs infected with Ad-Parkin, transfected with siRNAs, and treated with CCCP (10 µM). E , Schematics of generation of RBX2 and Parkin double knockout (RBX2 CKO /Parkin -/- ) mice. F , Ejection fraction and fractional shortening at 5 (Parkin -/- : n=5, RBX2 Het /Parkin +/- : n=8, RBX2 CKO : n=12. RBX2 CKO /Parkin -/- , n=8) and 8 (Parkin -/- : n=7, RBX2 Het /Parkin +/- : n=7, RBX2 CKO : n=11. RBX2 CKO /Parkin -/- , n=8) months of age. G , Survival curves of indicated mice. K , A proposed model showing the role of RBX2-CRL5 in regulation of physiological mitophagy and cardiac homeostasis. Student t test was used in A and B . One-way ANOVA followed by post hoc Tukey test in F . Log-rank (Mantel-Cox) test in G . ** P <0.01. ns, not significant.

Article Snippet: Cardiomyocyte-specific RBX2 knockout (RBX2 CKO ) mice were generated by crossing a Rbx2 Flox allele with Exon 1 flanked with loxP sites with αMHC Cre/+ mice (the Jackson Laboratory, strain #011038).

Techniques: Western Blot, Infection, Transfection, Isolation, Double Knockout

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Western blots of indicated proteins in NRVCs. Cells were infected with Ad-PINK1, transfected with indicated siRNAs and treated with or without CCCP (10 µM) for 12 hours. B , Western blots. RBX2 was deleted in Hela cells via CRISPR/Cas9 using a single guided RNA against RBX2 (gRBX2). WT and RBX2KO cells with treated with CCCP (10 µM) for indicated times before harvest. C , Analysis of Pink1 transcript levels in NRVCs transfected with indicated siRNAs by qPCR. D , Western blots. NRVCs were infected with Ad-PINK1, transfected with siRNAs, and treated with Bortezomib (BZM, 100 nM) for 6 hours. E , Cycloheximide-based pulse chase assay. NRVCs were transfected with siRNAs, treated with CCCP (10 µM) for 3 hours, followed by removal of CCCP, and then chased for the indicated time in the presence of cycloheximide (CHX, 100 nM). F , Immunoprecipitation of PINK1, followed by Western blots. NRVCs were transfected with indicated siRNAs and treated with or without Bortezomib (BZM, 100 nM) for 6 hours. G , Western blots of cell lysates from NRVCs transfected with indicated siRNAs and treated with CCCP (10 µM) for 3 hours. H , A proposed model showing the role of RBX2-CRL5 in regulation of physiological mitophagy and cardiac homeostasis.

Article Snippet: Cardiomyocyte-specific RBX2 knockout (RBX2 CKO ) mice were generated by crossing a Rbx2 Flox allele with Exon 1 flanked with loxP sites with αMHC Cre/+ mice (the Jackson Laboratory, strain #011038).

Techniques: Western Blot, Infection, Transfection, CRISPR, Pulse Chase, Immunoprecipitation