EMBO/soda-nel huggingface dataset:
ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress (2020)
Figure 3

Figure 3. CHK2 phosphorylates Beclin 1 at Ser90 and Ser93

(A) The indicated recombinant fragments of GST-Beclin1 were incubated with recombinant CHK2 in the presence of 32P-labeled ATP for in vitro kinase assays. MBP was used as a positive control. Ponceau staining indicates the expression of GST-Beclin 1 fragments for in vitro kinase assays. The asterisks in the blot indicate the domain of phosphorylated Beclin 1.

(B) Schematic representation of recombinant fragments of Beclin 1.

(C) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST-Beclin 1-WT, S9093A (AA), S90A and S93A protein. Reactions were analyzed by SDS-PAGE followed by autoradiography. The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants.

(D-E) Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation (D) or H2O2 (500μM) stimulation (E).

(F-G) Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation (F) or H2O2 (500μM) stimulation (G).

(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation.
ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress (2020)
Figure 4

Figure 4. CHK2-mediated Beclin 1 phosphorylation regulates autophagy

(A) The effect of Beclin 1 Ser90/93 phosphorylation on the interaction between Beclin 1 and Bcl-2. GST-Beclin1 WT, AA mutant and DD mutant (bait) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag-Bcl-2 (preys).

(B) Immunoprecipitation of Bcl-2 with Flag-Beclin 1 WT, AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 (500μM) treatment for 1h.

(C-D) Autophagy levels were determined in Beclin 1-depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant or DD mutant in normal medium or after H2O2 (500μM) treatment for 3h in the presence (C) or absence (D) of CHK2.

(E) Representative electron microscopic image of an autophagosome (arrow, left panel, AP) and an autolysosome (arrow, right panel, AL) in H1299 Beclin 1 WT cells treated with H2O2 (500μM) for 6 hr. Scale bars, 500 nm. Electron microscopic quantification of autophagosomes (AP) and autolysosomes (AL) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 (500μM) treatment. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.001 compared to Beclin 1 WT; #P < 0.001 compared to Beclin 1 WT treated with H2O2; &P < 0.001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2.

(F) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93, Beclin 1, p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment for 3h.
ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress (2020)
Figure 5

Figure 5. ROS acts as a signaling molecule to activate the CHK2-Beclin 1 axis under glucose starvation and hypoxic stress

(A) Western blot detection of p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.

(B) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.

(C) Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.

(D) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.

(E) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to hypoxia with or without pretreatment with NAC.

(F) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to glucose starvation with or without pretreatment with NAC.

(G) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, and AMPKα in H1299 cells in normal medium or after H2O2 (25μM) stimulation.

(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG (5mM) and oligomycin (2.5μM) treatment.
ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress (2020)
Figure 6

Figure 6. CHK2-mediated autophagy limits ROS levels by clearing damaged mitochondria

(A-B) Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation (Glu-) (A) or 36hr hypoxia (B).

(C) Western blot detection of CHK2, HSP60, TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation (Glu-) or 36 hr hypoxia.

(D-E) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation (Glu-) medium or hypoxia. The yellow asterisks mark mitochondria. The red arrows indicate autophagic structures. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ***P < 0.001 compared to shNC treated with H2O2 or hypoxia (Mann-Whitney test); Scale bar, 1 μm.

(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm. Scale bar, 10 μm.

(G-H) The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation (G) or 48 hr hypoxia (H). All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).
ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress (2020)
Figure 7

Figure 7. CHK2-mediated autophagy protects against cell death and tissue damage following cerebral ischemia

(A-B) H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation (A) or 48h hypoxia stimulation (B). Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments; *p < 0.05 compared to NC glucose starvation (A); **p < 0.01 compared to NC hypoxia (B) (Student's t-test).

(C-D) H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation (C) or 48h hypoxia stimulation (D). Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).

(E) CHK2+/+ and CHK2-/- mice were subjected to MCAO for 1 hr and reperfusion for 12 h. Contralateral (C) and ipsilateral (I) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC.

(F) The infarct volume was determined by measuring infarct size relative to normal in the slice. (n = 5 mice). Data are presented as mean ± s.e.m.; **P < 0.01 (Student's t-test).

(G-J) Immunoblots for p62, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion-treated CHK2+/+ and CHK2-/- mice. Quantification of p62, p-Beclin 1 Ser90 and p-Beclin 1 Ser93 protein levels (n = 3 mice). Data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01 (Student's t-test).

Expanded View Figure legends
Regulation of cohesin-mediated chromosome folding by PDS5 in mammals (2022)
Figure 1

Figure 1. Acute PDS5A or PDS5B degradation with the inducible dTAG technology.

A Schematic of the generation of PSD5A-dTAG or PDS5B-dTAG PLC/PRF/5 cells.

B Western blots of whole-cell extracts showing efficient degradation of PDS5A or PDS5B (left, right) with dTAG treatment (at different time points).

C Genome browser track at a defined region of chromosome 1 for Flag-PDS5A, PDS5A in PDS5A-dTAG cells treated with DMSO or dTAG for 24hr (left), or Flag-PDS5B, PDS5B in PDS5B-dTAG cells treated with DMSO or dTAG for 24hr (right).

D ChIP-seq signal for FLAG-PDS5A and FLAG-PDSB (ChIP assays were performed with antibodies against Flag and PDS5A/B) centered at TSS of promoters (± 3 kb) across all peaks called for each of the proteins in DMSO treated PDS5A-dTAG or PDS5B-dTAG cells, showing the drastic reduction in PDS5A/B enrichment on chromatin after degradation. Top: normalized signal intensities for each protein.

E Representative track examples of small genomic regions showing complete depletion of PDSA5A and PDS5B after dTAG treatment (left, right).

F ChIP-qPCR using Flag antibody demonstrating complete depletion of PDS5A or PDS5B (left, right) in H19/IGF2 imprinting locus and the promoter region of PCDH10, NC represents a negative binding site. Data are represented as mean±SD from three biological replicates.
Regulation of cohesin-mediated chromosome folding by PDS5 in mammals (2022)
Figure 2

Figure 2. Similar genomic distribution of PDS5A and PDS5B with cohesin and CTCF.

A Genome browser track showing genomic distribution of FLAG-PDS5A, FLAG-PDS5B, RAD21, and CTCF at a selected region of chromosome 2. FLAG-PDS5A and FLAG-PDS5B ChIP-seq experiments were performed using Flag antibody from PDS5A-dTAG or PDS5B-dTAG cells mock depleted of PDS5A or PDS5B. ChIP-seq data of RAD21 and CTCF were produced from PDS5A-dTAG cells without dTAG treatment.

B Heatmaps of signal intensities for FLAG-PDS5A, FLAG-PDS5B, CTCF, and RAD21 ChIP-seq data across all defined FLAG-PDS5A peaks.

C Averaged ChIP-seq signal densities of FLAG-PDS5A, FLAG-PDS5B, RAD21, and CTCF on PDS5A & PDS5B common sites and PDS5A/PDS5B unique sites.

D Veen Diagram analysis depicting the overlapping binding events of FLAG-PDS5A or FLAG-PDS5B with RAD21 and CTCF, or between FLAG-PDS5A and FLAG-PDS5B.

E Averaged signal plots displaying the distribution of PDS5A, PDS5B, RAD21 and CTCF at DNA loop anchors and TAD boundaries defined with following Hi-C data of PDS5A-dTAG cells without dTAG treatment.

F Reciprocal Re-ChIP for PDS5A and PDS5B. The co-binding of PDS5A and PDS5B at PDS5A&PDS5B common sites were assayed by Re-ChIP-qPCR. The second ChIP for Re-ChIP was performed using antibodies against PDS5A, FLAG(PDS5B), RAD21, control IgG. RAD21 served as a positive control. Data are shown as fold enrichment versus IgG (mean±SD from three biological replicates).

G Heatmaps and averaged signal plots showing the differential PDS5B or PDS5A distribution on sites defined in Fig 1B after PDS5A or PDS5B depletion.
Regulation of cohesin-mediated chromosome folding by PDS5 in mammals (2022)
Figure 5

Figure 5. Co-depletion of PDS5A and PDS5B reduces the levels of cohesin at loop anchors

A Validation of PDS5A;PDS5B-dTAG cells for simultaneous depletion of PDS5A and PDS5B.

B Chromatin fractionation assay showing the change of chromatin-bound level of RAD21 or CTCF after co-depletion of PDS5A and PDS5B.

C Heatmaps displaying the ChIP-seq binding intensities for significantly changed RAD21 peaks after joint PDS5 proteins depletion (left). Veen diagram showing RAD21 peaks identified in cells before and after co-depletion of PDS5A and PDS5B.

D Changes of RAD21 signals at all loop anchors defined in PDS5A-dTAG cells or PDS5B-dTAG cells with DMSO treatment after PDS5A and PDS5B co-depletion, or either depletion. For PDS5A;PDS5B-dTAG cells, we used the genomic positions of loop anchors identified in DMSO treated PDS5A-dTAG cells.

E Differential RAD21 ChIP-seq signals of representative regions showing significantly down-regulated peaks after co-depletion of PDS5A and PDS5B.

F Differential RAD21 ChIP-seq signals of representative regions showing significantly up-regulated peaks after co-depletion of PDS5A and PDS5B.
Fibroblast state switching orchestrates dermal maturation and wound healing (2018)
Figure 1

Fig 1. Dermal architecture is defined by an inverse correlation between fibroblast proliferation and ECM deposition.

(A) Quantification of fibroblast density (number of PDGFRαH2BEGFP+ cells, right; n=3 biological replicates per time point) and dermis volume (left) with age (n=7 E12.5; n=8 P0, P6; n=9 E16.5, E17.5, P4; n=10 P10, P50; n=11 E10.5; n=12 E18.5, P2, P21; n=20 P12 biological replicates). (B) Changes in fibroblast proliferation and collagen deposition during dermal development in mice. Immunofluorescence staining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin at indicated developmental time points (upper panel). Polarised light images of Picrosirius red stained back skin sections shown in binary images at indicated time points (lower panel). (C) Percentage of PDGFRαH2BEGFP+ cells in G1 cell cycle phase with age (n=1 E12.5; n=2 P2, P10; n=3 E16.5, E17, P0; n=4 E10.5; n=5 E17.5, E18.5, P50; n=7 P4 biological replicates) fitted using an exponential model. (D) Quantification of fibroblast proliferation (left; n= 4 E16.5, P10; n= 3 E10.5, E18.5, P2, P21, P44, P50; n= 2 E17.5, P0 biological replicates) and collagen density (right; n= 4 E16.5, P2, P6, P10, P14, P16, P21, P50; n= 3 E10.5, E18.5, P2, P21, P44, P50 biological replicates) with age. (E) Immunofluorescence image of R26Fucci2a x Dermo1Cre back skin at P2 where mVenus-hGem (green) expressing cells are in S/G2/M phase and mCherry-hCdt1 (red) positive cells are arrested in G1 phase. (F) Percentage of labelled cells in S/G2/M cell cycle phase in the upper and lower dermis at P0 (n=3 biological replicates) and P2 (n=4 biological replicates). (G-I) Changes in dermal cell proliferation and collagen deposition in human skin. (G) Immunofluorescence staining for Vimentin (red) and Ki67 (green) of back skin at indicated time points (upper panel). Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points (lower panel). (H) Quantification of proliferating cells in the upper and lower dermis (n=3 biological replicates per time point). (I) Quantification of dermal cell proliferation (Ki67+) (left) and collagen density (right) with age (n= 3 biological replicates per time point). Note that cells in upper human dermis were more proliferative than cells in the lower dermis at all developmental time points analysed and that fibroblasts entered a quiescent, non-proliferative state before collagen was efficiently deposited. Data shown are means ±s.d. Nuclei were labelled with DAPI (blue in B,E,G). Scale bars, 100µm. wk, week.
Fibroblast state switching orchestrates dermal maturation and wound healing (2018)
Figure 2

Fig 2: ECM triggers fibroblast state switching

(A) Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in (triangle) and outside (circle) collagen gels for up to 5 days. Cell number was measured by CellTiterGlo assay at indicated time points (n= 3 technical replicates of one representative experiment repeated 3 times). (B) Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days (triangle). Controls were kept on TCP for 3 days before plating (circle). Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown (n= 3 technical replicates). (C) Experimental design of de-epithelialized dermal (DED) organotypic cultures for reconstitution (left panel) and ECM degradation (right panel). (D,E) Repopulation of DED organotypic cultures recapitulates adult dermal homeostasis. (D) Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures with and without fibroblasts at indicated time points after seeding. (D) Quantification of Ki67 positive fibroblasts at indicated time point after seeding (n= 8 section per condition of one representative experiment repeated two times). (E,F) ECM degradation in DED organotypic cultures by collagenase treatment. (E) Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures at indicated time points after collagenase treatment. (F) Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment (n= 8 section per condition of one representative experiment repeated two times). Note that fibroblasts around the injection sides start to proliferate after 48 hours. Nuclei were labelled with DAPI (blue) (D,F). Scale bars, 100µm (D,F). Data shown are means ± s.d. Fb, fibroblasts; PD, post digestion; TCP, tissue culture plastic; h, hours.
Fibroblast state switching orchestrates dermal maturation and wound healing (2018)
Figure 4

Fig 4: Development of a 3D tissue model and in vivo live imaging during dermal maturation.

(A) (x,y) cross-section of the computational model simulation of the dermal maturation process at the indicated Monte Carlo Steps (MCS). Colour code indicates epidermis (green), proliferating fibroblasts (blue), quiescent fibroblasts (yellow), ECM (grey) and adipocytes (brown). See accompanying Movie EV1. (B) Abundance of the cell populations during the course of the simulation. (C) Quantification of fibroblast proliferation and ECM density in the simulation. (D) Live imaging of adult PDGFRαH2BEGFP mouse back skin. Representative time-lapse images of adult PDGFRαH2BEGFP (green) dermis with collagen shown as second harmonic generation (SHG) in purple at indicated imaging time points. Scale bar, 100 µm. See accompanying Movie EV2. (E-G) In silico lineage tracing of fibroblasts. (E) Labelling of fibroblasts close to the epidermis at indicated MCS. (F) Labelling of fibroblasts in the middle of the dermis at indicated MCS. (G) Labelling of fibroblasts close to the inside of the body at indicated MCS.
Fibroblast state switching orchestrates dermal maturation and wound healing (2018)
Figure 5

Fig 5: Fibroblast proliferation and ECM changes during wound healing.

(A,B) Experimental design of wound healing time course analysis. (A) Wound healing time course. Skin was collected at the indicated time points. (B) Quantification strategy is schematically illustrated. Red boxes upper dermis; black boxes lower dermis; solid lines inside and dashed lines outside the wound site. (C,D) Fibroblast activation during wound repair. Immunofluorescence staining for α−sma (red) of PDGFRαH2BEGFP (green) wound sites at indicated post wounding (PW) times (C) and quantification of α−sma fluorescence intensity in pixel per area inside the upper and lower wound bed (n= 4 PW0, PW2, PW7, PW21; n= 3 PW10, PW14; n= 2 PW4 biological replicates) (D). (E-H) Changes in fibroblast density, proliferation and collagen deposition during wound repair. (E) Immunostaining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin sections at indicated wound healing time points. (F) Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates). (G) Quantification of fibroblast density during wound repair inside and outside the wound bed (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates). (H) Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding. (I) Quantification of proliferating (Ki67 positive) keratinocytes and fibroblasts during wound repair over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates). Nuclei were labelled with DAPI (blue) (C,E). Scale bars, 100µm (C,E). WB, wound bed.
Fibroblast state switching orchestrates dermal maturation and wound healing (2018)
Figure 7

Fig 7: 3D model and in vivo live imaging of dermal wound repair.

(A) (x,y) cross-section of the computational simulation of wound healing in adult dermis. Please refer to associated Movie EV4. (B-D) Live imaging of adult PDGFRαH2BEGFP back skin during wound healing. (B) Experimental design of in vivo imaging of wound healing. (C) Representative wound bed Z-stack stitched maximum projections of the entire wound bed (upper panel) showing fibroblasts (green) and collagen with second harmonic generation (SHG) in purple at indicated time points after wounding and before live imaging. Dotted line indicates wound edge and boxed area indicates the position of the magnified area below. (D) Representative time-lapse images of adult wound bed at PW4, where Z-directional movement is visualized by pseudo colouring the fibroblast nuclei (red, movement towards the basement membrane; blue, movement away from the basement membrane). Dotted line indicates wound edge. Please refer to associated Movie EV6 which shows fibroblasts actively migrating into the wound bed. (E-H) In vivo lineage tracing of upper (Blimp1Cre) and lower (Dlk1CreER) dermal fibroblasts during wound healing. (E) Experimental strategy for in vivo lineage tracing. (F) Immunofluorescence image of Blimp1Cre x Confetti (upper panels) and Dlk1CreER x Confetti (lower panel) back skin inside and outside the wound bed at 10 days post wounding. Colours show YFP (green), RFP (red) and CFP (blue) with bright field image overlaid. (G) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding (n= 400 clones of 4 biological replicates). (H) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding (n=300 clones of 2 biological replicates). Scale bars, 500µm (C); 100µm (D,F).
Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere (2021)
Figure 2

Figure 2. MuRF1-mediated ubiquitination of the kinase region of titin

A Anti-TK Western blot of ubiquitinated titin samples.

B Reconstruction of the 3D-structure of the full-length kinase region of titin, A168-M1, achieved by overlapping the structure of A170-M1 in this work and that of the A168-A170 previous reported (PDB 2NZI). The presence of a flexible hinge region at the A170-NL junction can be predicted since the crystal structure of A170-M1 shows only few and weak interactions between A170 and TK. Otherwise, the A168-M1 titin segment is highly structured. Lysine residues ubiquitinated by MuRF1 (as identified by mass spectrometry) are marked in magenta. Sites found in denervated limb muscle from mice are boxed (Lang et al, 2017) and sites found in other in vivo studies are underlined (Wagner et al, 2012).
Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere (2021)
Figure 3

Figure 3: The cellular colocalization of titin A168-TK with Nbr1 and p62 is MuRF1 dependent

A Domain composition of Nbr1 and p62 proteins, where PB1: Phox and Bem1; ZZ: ZZ finger; NBR1: Next to BRAC1; LIR: light-chain-3-interacting region; UBA: ubiquitin-binding domain. The ubiquitin binding function of the UBA domain is indicated by an open triangle labeled "Ub".

(B-D) Micrographs of H9C2 cells corresponding to (B) expressions of single species: MuRF1-mCherry (red), titin EGFP-A168-TK (green), Venus-Nbr1 (yellow) or Cerulean-p62 (cyan); (C) Co-expression of titin EGFP-A168-TK with mCherry-MuRF1. Neither Nbr1 nor p62 were co-transfected. The titin fragment and MuRF1 co-localize forming filamentous formations, which is in agreement with their known complexation; (D) Co-expression of MuRF1-mCherry (red), titin EGFP-A168-TK (green) and Venus-Nbr1 (yellow) or Cerulean-p62 (cyan) or respective truncated variants of the latter lacking the ubiquitin binding domain (∆UBA). (Throughout, cell nuclei are stained with DAPI; blue). (Scale bar is 10 μm in all cases).