anti rbx2  (Proteintech)

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , Representative image sequence demonstrating cGAS chromosome attachment in mitosis followed by intranuclear redistribution and degradation in cGAS–GFP-expressing HeLa cells (Supplementary Video ). Scale bars, 10 μm. b , The relative nuclear cGAS–GFP mean fluorescence intensity (MFI) in post-mitotic HeLa cells. n = 29. c , QIBC analysis of endogenous nuclear (left) and cytosolic (right) cGAS levels in HeLa cells. d , The relative nuclear cGAS–GFP MFI in post-mitotic HeLa cells that were treated with epoxomicin ( n = 31), bortezomib ( n = 30) or DMSO ( n = 31). e , Focused RNAi-based screen of UPS factors regulating nuclear cGAS–GFP abundance. Each dot shows the cGAS–GFP mean intensity blotted against integrated intensity. Knockdowns of genes that yielded increased cGAS–GFP levels (>3 s.d.) relative to the control siRNA are shown in yellow. Genes encoding cGAS (dark blue), proteasomal subunits (light blue), CRL5 complex components (dark green) and SPSB3 (light green) are highlighted. f , Schematic of CRL5–SPSB3-directed cGAS ubiquitylation. g , The relative nuclear cGAS–GFP MFI in post-mitotic HeLa cells treated with siRNA against SPSB3 (siS PSB3 ; n = 18) or CUL5 ( n = 14), non-targeting control siRNA ( n = 16) or epoxomicin ( n = 14). h , QIBC analysis of nuclear cGAS levels in HeLa cells that were treated with siRNA against SPSB3 or CUL5 or control siRNA. i , j , Nuclear (Nuc.) and cytosolic (Cyto.) cGAS–GFP was analysed using immunoblotting in cGAS–GFP-expressing HeLa cells that were treated with siRNA against SPSB3 or control siRNA ( i ) or MLN4924 ( j ) and incubated with cycloheximide (CHX) for the indicated time courses. H2B and GAPDH were used as the loading controls. For b , d and g , data are mean ± s.d. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Šídák’s multiple-comparison test ( c and h ) and two-way ANOVA with Tukey’s multiple-comparison test ( d and g ). For a , i and j , one representative experiment of three independent experiments is shown.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Sequencing, Expressing, Fluorescence, Western Blot, Incubation, Comparison

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , Results from the siRNA screen highlighting cGAS-GFP nuclear abundance by mean fluorescence intensity and integrated fluorescence intensity in cells treated with siRNAs against SPSB family members ( n = 3). b , Nuclear cGAS measurements by confocal microscopy in HeLa cells (endogenous cGAS) or U2OS cells (cGAS-GFP expression) transfected with siRNAs against CUL5 , SPSB3 and PSMA5 or control siRNA or treated with epoxomicin or DMSO ( n = 2). c , Relative nuclear cGAS-GFP MFI measurement in post-mitotic U2OS cells treated with siRNA against SPSB3 ( n = 15) or CUL5 ( n = 15), control siRNA ( n = 14) or epoxomicin ( n = 15). d , Nuclear cGAS-GFP measurements by confocal microscopy in HeLa cells treated with siRNA against SPSB3 ( n = 31) or CUL5 ( n = 33) or control siRNA ( n = 22). e , Whole cell lysates, cytosolic and nuclear fractions were extracted from HeLa cells transfected with non-targeting control siRNA of siRNAs targeting SPSB3 or CUL5 and analysed by immunoblot. Vinculin and H2B were used as loading control of whole cell lysates, cytosolic and nuclear fractions, respectively. f , mRNA levels of cGAS were measured by RT-qPCR in HeLa cells treated with non-targeting control siRNA or siRNAs against SPSB3 or CUL5 for 5 days. Ratios of relative cGAS mRNA levels normalized to the control are shown ( n = 3). g , QIBC analysis of endogenous cytosolic cGAS level in HeLa cells treated with siRNA against SPSB3 , CUL5 or control siRNA. h , Nuclear cGAS-GFP measurements by confocal microscopy in HeLa cells treated with epoxomicin ( n = 32), MLN4924 ( n = 29), or DMSO ( n = 32). i , QIBC analysis of endogenous cGAS levels in HeLa cells treated with MLN4924 or DMSO. j , mRNA levels of cGAS were measured by RT-qPCR in HeLa cells treated with MLN4924 or DMSO. Ratios of relative cGAS mRNA levels normalized to the control are shown ( n = 6). k , Cytosolic and nuclear fractions were extracted from HeLa cells expressing doxycycline (Dox)-inducible SPSB3 that were treated or not with Dox for 4 days). Immunoblots probing cGAS and SPSB3 (FLAG) are shown. Vinculin and H2B were used as loading control for cytosolic and nuclear fractions, respectively. l , Whole cell lysates and nuclear fractions collected from primary endothelial cells, BJ-5ta fibroblasts or differentiated THP-1 cells treated with non-targeting control siRNA or siRNA against SPSB3 for 5 days were analysed by immunoblot. Vinculin and H2B were used as loading control for whole cell lysates and nuclear fractions, respectively. Numbers indicate individual cells ( c , d , h ) or technical replicates( a , f , j ). Data are mean ± SD ( a , c , d , f , g , h , j ) or mean ± SEM ( i ). P values were obtained by two-way ANOVA with Tukey’s multiple comparison test ( c ), one-way ANOVA with Dunnett’s multiple comparison test ( d , h ), one-way ANOVA ( f ), one-way ANOVA with Šídák’s multiple comparison test ( g ) or two-tailed Student’s t -test ( j ). One representative of three ( e , f , k - l ) independent experiments is shown.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Fluorescence, Confocal Microscopy, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Comparison, Two Tailed Test

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , IP of Flag-tagged cGAS from HEK293T cells transfected with cGAS-Flag and HA-ubiquitin and treated with indicated siRNAs. Samples were analysed by immunoblot. Vinculin was used as a loading control. b , c , Representative confocal microscopy images of HeLa cells stained for SPSB3 ( b ) or CUL5 ( c ) and DAPI (blue). Scale bars, 20 μm. d , QIBC analysis of endogenous nuclear (left) or cytosolic (right) SPSB3 levels in HeLa cells. e , IP of GFP-tagged NLS-cGAS from HEK293T cells transfected with constructs for GFP-tagged NLS-cGAS, HA-tagged ubiquitin with or without SPSB3. Samples were analysed by immunoblot. f , Size-exclusion chromatography (SEC) and SDS–PAGE of an assembled Halo-cGAS-SPSB3-ELOBC complex. g , SEC and SDS–PAGE of an assembled cGAS-SPSB3-ELOBC complex. Data are mean ± SD ( d ). P values were obtained by one-way ANOVA with Šídák’s multiple comparison test ( d ). One representative of three ( a - d ) or two ( e - g ) independent experiments is shown.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Transfection, Western Blot, Confocal Microscopy, Staining, Construct, Size-exclusion Chromatography, SDS Page, Comparison

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , A composite cryo-EM density map of the nucleosome–cGAS–SPSB3–ELOBC complex, assembled from two focused-refinement maps (nucleosome–cGAS and cGAS–SPSB3–ELOBC). Different contour levels were used for optimal visualization using UCSF ChimeraX . cGAS(site C), mutated in the DNA-binding C site, was used to obtain this dataset. b , Ribbon representation of the nucleosome–cGAS–SPSB3–ELOBC complex structure. The arrows indicate the B30.2/SPRY and SOCS box domains of SPSB3. c , Detailed view of the binding interface between cGAS and SPSB3. d , Sequence logo analysis of the cGAS and SPSB3 interface derived from 150 vertebrate species. e , Model of nucleosome-bound cGAS targeted by an activated (neddylated) CRL5–SPSB3 complex with RBR E3 ligase ARIH2 priming polyubiquitylation by transferring the first ubiquitin onto cGAS. Lysine residues that have been identified as cGAS ubiquitylation sites by MS and are essential for SPSB3-mediated degradation of cGAS are coloured red; the catalytic Cys310 of ARIH2 is coloured gold. The model was built by docking the nucleosome–cGAS–SPSB3–ELOBC model into a model comprising the ELOBC–CUL5 (Protein Data Bank (PDB): 4JGH ) , CUL5–NEDD8–RBX2–ARIH2 (PDB: 7ONI ) and ARIH1–UB (PDB: 7B5M ) complexes.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Cryo-EM Sample Prep, Binding Assay, Sequencing, Derivative Assay, Transferring

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , Right: Ribbon diagram and a 3D reconstruction of the nucleosome-cGAS siteC -SPSB3-ELOBC complex. cGAS in blue, SPSB3 in green, nucleosomal DNA in lilac, histones in grey, ELOB in yellow, ELOC in antique white. Left: EM densities (shown as mesh at 5σ) for cGAS residues interacting with the SPSB3. Shown are cGAS N513, N514 and R512/D465 interactions with SPSB3 respectively. b , The cGASSPSB3 model is aligned with the VASA-SPSB1 model (PDB: 3F2O) , individual interacting residues are shown by sticks. c , Sequence alignment of SPSB1/2/3/4. Residues used for substrate recognition are highlighted. d , Percent identity matrix of human SPSB1/2/3/4. e , Model of the nucleosome/cGAS 2:2 sandwich targeted by an activated (neddylated) CRL5-SPSB3 complex with RBR E3 ligase ARIH2 priming polyubiquitylation by transferring the first ubiquitin onto cGAS. cGAS K427/K428 is competitively targeted by either nucleosomal DNA or the E3 ligase. The model is built by docking the nucleosome-cGAS-SPSB3-ELOBC model into a model composed of the nucleosome/cGAS 2:2 complex (PDB: 6Y5D) , ELOBC-CUL5 (PDB: 4JGH) , CUL5-NEDD8-RBX2-ARIH2 (PDB: 7ONI) , and ARIH1-UB (PDB: 7B5M) complexes.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Sequencing, Transferring

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , mRNA levels of cGAS were measured by RT-qPCR in HeLa cGAS KO cells reconstituted with cGAS (WT), cGAS(NN), or cGAS(KK). Ratios relative to WT levels are shown ( n = 3). b , Expression levels of cGAS , ISG15 or IFNB1 were assessed by RT-qPCR in HeLa cGAS KO cells reconstituted with doxycycline-inducible cGAS (WT) or a cGAS mutant defective in DNA binding and ubiquitylation (KRKKNN (K173E/R176E/K407E/K411A/N513/514A)) after 4 days of doxycycline treatment. Ratios of relative cGAS , ISG15 and IFNB1 mRNA levels normalized to the control are shown ( n = 3). c , Cells from ( b ) were lysed and analysed by immunblot for cGAS and ISG15 levels. Vinculin was used as a loading control. d , cGAS and ISG15 measurements by immunoblot in HeLa cGAS KO cells transfected with mRNA of encoding for human cGAS or cGAS N513A/N514A (NN). Cells were harvested on day 0, 1, 2, or 4 after transfection. Vinculin was used as a loading control. e , cGAS and ISG15 measurements by immunoblot in CT26 cGas KO cells transfected with mRNA of encoding for mouse cGas or cGAS N498A/N499A (NN). Cells were harvested on day 0, 1, 2, or 4 after transfection. Vinculin was used as a loading control. f , Expression of SPSB3 , ISG15 or IFNB1 were assessed by RT-qPCR 3 days after enforced expression or not of SPSB3 with doxycycline in HeLa cells. Ratios of relative SPSB3 , ISG15 and IFNB1 mRNA levels normalized to the control are shown ( SPSB3 , n = 3; ISG15 , IFNB1 , n = 4). g - i , mRNA expression levels of genes as indicated were assessed by RT-qPCR in primary endothelial cells ( n = 3) ( g ), BJ-5ta fibroblast cells ( n = 3) ( h ), and differentiated THP-1 cells ( n = 3) or THP-1 cGAS KO cells ( n = 3) ( i ) treated with non-targeting control siRNAs or siRNAs against SPSB3 for 5 days. Ratios of the relative expression of each transcript compared to the controls are shown. j , Induction of IFNB1 mRNA was measured by RT-qPCR in HeLa cGAS KO cells reconstituted with cGAS (WT), cGAS(NN), or cGAS(KK) infected with HSV-1 (MOI = 0.1) or VACV (MOI = 1) for 18 h ( n = 4). k , HeLa cells treated with non-targeting control siRNAs or siRNAs against SPSB3 or CUL5 for 5 days were infected with HSV-1-GFP (left, MOI = 0.01) or VACV-GFP (right, MOI = 0.5) for 24 h ( n = 3). GFP + cells were quantified by flow cytometry. Data are mean ± SD, numbers indicate the number of technical replicates ( a - b , f - k ). P values were obtained by one-way ANOVA ( a , f , g - k ) or two-tailed Student’s t -test ( b , f ). One representative of two ( a ) or three ( b - k ) independent experiments is shown.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Binding Assay, Western Blot, Transfection, Infection, Flow Cytometry, Two Tailed Test

Journal: Cell reports

Article Title: The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation

doi: 10.1016/j.celrep.2024.115101

Figure Lengend Snippet: (A) Ablation of RBX1 diminishes amino acid-induced mTORC1 activation in EMSCs. Three gRNAs designed to target distinct exon regions of RBX1 were used. Cells were starved in amino acid-deprived medium for 50 min and treated with 1× amino acids for 10 min. gRNA targeting GFP was used as a control. Note that RBX1–3 gRNA failed to ablate RBX1. (B) Ablation of RBX1, but not RBX2, diminishes amino acid-induced mTORC1 activation in EMSCs. Cells were treated as in (A). Levels of phosphorylated S6K1 in the indicated cells (amino acid-replete conditions) were quantified and expressed as the ratio of pS6K/S6K1. * p < 0.05, ** p < 0.01, mean ± SD, n = 4. (C) Ablation of RBX1 diminishes CQ/MG-induced mTORC1 activation in Ragulator-deficient cells. RBX1 is ablated in p18/LAMTOR1 knockout (KO) EMSCs. Cells were treated with CQ (50 μM) or/and MG (20 μM) in amino acid-free medium for 45 min. (D) Ablation of RBX1 diminishes Rheb ubiquitination. RBX1 KO EMSCs were lysed under amino acid-replete conditions in RIPA buffer containing the deubiquitinase inhibitor (100 mM) N-ethylmaleimide (NEM). The lysates were subjected to SDS-PAGE, and levels of Rheb and Ub-Rheb were monitored with a Rheb antibody. Non-Ub-Rheb and Ub-Rheb are indicated. (E) MLN4924 specifically inhibits mTORC1 activity induced by amino acids in EMSCs. Cells were treated with the indicated concentrations of MLN4924 for 16 h. Cells were amino acid starved for 50 min and re-stimulated with 1× amino acids for 10 min. Levels of phosphorylated S6K1 in the indicated cells (amino acid-replete conditions) were quantified and expressed as the ratio of pS6K/S6K1. **** p < 0.0001, mean ± SD, n = 4. (F) MLN4924 inhibits mTORC1 activity induced by amino acids in both control and DEPDC5 KO HEK293T cells. Cells were treated with MLN4924 (1 μM) as in (E). Cells were amino acid starved for 50 min and re-stimulated with 1× amino acids for 10 min. The effect of DEPDC5 ablation was confirmed by the high levels of mTORC1 activity resistant to amino acid starvation. (G) MLN4924 diminishes levels of Ub-Rheb in HEK293T cells. Cells were treated with MLN4924 (1 μM) for 16 h and lysed as in (D), and the lysates were subjected to immunoblotting with a Rheb antibody.

Article Snippet: RBX2 polyclonal antibody , Proteintech , Cat# 11905-1-AP; RRID:AB_10697836.

Techniques: Activation Assay, Control, Knock-Out, Ubiquitin Proteomics, SDS Page, Activity Assay, Western Blot

Journal: Cell reports

Article Title: The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation

doi: 10.1016/j.celrep.2024.115101

Figure Lengend Snippet:

Article Snippet: RBX2 polyclonal antibody , Proteintech , Cat# 11905-1-AP; RRID:AB_10697836.

Techniques: Virus, Recombinant, Magnetic Beads, Control, Negative Control, Ubiquitin Proteomics, shRNA, Plasmid Preparation, Immunofluorescence, Software

Journal: iScience

Article Title: Discovery of SOCS7 as a versatile E3 ligase for protein-based degraders

doi: 10.1016/j.isci.2024.109802

Figure Lengend Snippet: Description of the E3 ligases used in the protein-based degrader screening

Article Snippet: RBX2 rabbit antibody , Abcam , Cat#ab181986.

Techniques:

Journal: iScience

Article Title: Discovery of SOCS7 as a versatile E3 ligase for protein-based degraders

doi: 10.1016/j.isci.2024.109802

Figure Lengend Snippet:

Article Snippet: RBX2 rabbit antibody , Abcam , Cat#ab181986.

Techniques: Recombinant, Transfection, Magnetic Beads, Fluorescence, Expressing, Software, Sterility, Suction Filtration

Journal: iScience

Article Title: Discovery of SOCS7 as a versatile E3 ligase for protein-based degraders

doi: 10.1016/j.isci.2024.109802

Figure Lengend Snippet: Description of the E3 ligases used in the protein-based degrader screening

Article Snippet: Primary antibodies include anti-GFP (1/1000, Novus, Cat#NB100-1770), anti-FLAG (1/2000, Sigma, Cat#F1804), anti-actin (1/50000, Sigma, Cat#MAB1501), anti-α-tubulin (1/2000, Abcam, Cat#ab4074), anti-HSP90 (1/4000, CST, Cat#4874S), anti-KRAS (1/100, Santa Cruz Biotechnologies, Cat#sc-30), anti-NRAS (1/1000, Abcam, Cat#ab77392), anti-HRAS (1/500, Proteintech, Cat#18295-1-AP), anti-ALFA HRP-linked (1/4000, NanoTag Biotechnologies, Cat#N1505), anti-Elongin-C (1/1000, Abcam, Cat#ab226831), anti-Elongin-B (1/1000, Abcam, Cat#ab151743), anti-Cullin 5 (1/1000, Abcam, Cat#ab184177), anti-RBX2 (1/1000, Abcam, Cat#ab181986), anti-phospho-p44/22 MAPK (ERK1/2) (1/1000, CST, Cat#4370S), anti-p44/42 MAPK (total ERK1/2) (1/1000, CST, Cat#4695S), anti-phospho-AKT S473 (1/1000, CST, Cat#4058S) and anti-AKT (1/1000, CST, Cat#9272S).

Techniques:

Journal: iScience

Article Title: Discovery of SOCS7 as a versatile E3 ligase for protein-based degraders

doi: 10.1016/j.isci.2024.109802

Figure Lengend Snippet:

Article Snippet: Primary antibodies include anti-GFP (1/1000, Novus, Cat#NB100-1770), anti-FLAG (1/2000, Sigma, Cat#F1804), anti-actin (1/50000, Sigma, Cat#MAB1501), anti-α-tubulin (1/2000, Abcam, Cat#ab4074), anti-HSP90 (1/4000, CST, Cat#4874S), anti-KRAS (1/100, Santa Cruz Biotechnologies, Cat#sc-30), anti-NRAS (1/1000, Abcam, Cat#ab77392), anti-HRAS (1/500, Proteintech, Cat#18295-1-AP), anti-ALFA HRP-linked (1/4000, NanoTag Biotechnologies, Cat#N1505), anti-Elongin-C (1/1000, Abcam, Cat#ab226831), anti-Elongin-B (1/1000, Abcam, Cat#ab151743), anti-Cullin 5 (1/1000, Abcam, Cat#ab184177), anti-RBX2 (1/1000, Abcam, Cat#ab181986), anti-phospho-p44/22 MAPK (ERK1/2) (1/1000, CST, Cat#4370S), anti-p44/42 MAPK (total ERK1/2) (1/1000, CST, Cat#4695S), anti-phospho-AKT S473 (1/1000, CST, Cat#4058S) and anti-AKT (1/1000, CST, Cat#9272S).

Techniques: Recombinant, Transfection, Magnetic Beads, Fluorescence, Expressing, Software, Sterility, Suction Filtration

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

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: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

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: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

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: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

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: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

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: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

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: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

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