EMBO/SourceData huggingface dataset:
Figure legend: "Overexpression of DBN1, DBN1S142A and DBN1S142D in RPE1 WT cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. ** P≤ 0.01; *** P≤ 0.001; ns: not specific."
NCBI gene IDs:
"DBN1: 1627"
Figure legend: " The phospho-inhibitory (S142A) version of DBN1 rescued the elongated cilia of DBN1 KO cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. * P≤ 0.05; *** P≤ 0.001; ns: not significant. Pairwise comparison was done with unpaired student's t-test and the p-value was written in the figure. Global comparison was done with one-way ANOVA and the p-values were marked with stars. "
NCBI gene IDs:
"DBN1: 1627"
Figure legend: " The overexpressed WT DBN1 was hyper-phosphorylated in cells as detected by phospho-specific DBN1pS142 antibodies. The DBN1pS142 antibodies did not detect the overexpressed DBN1S142A or DBN1S142D. "
NCBI gene IDs:
"DBN1: 1627"
Figure legend: " Immunoblot analysis confirming the successful siRNA depletion of CDK5 and indicating hypo-phosphorylation of DBN1S142 in response to CDK5 depletion The DBN1pS142/GAPDH ratio from (H) was densitometrically measured. Three independent experiments. Mean ± SD. * P≤ 0.05 "
NCBI gene IDs:
"CDK5: 1020"
Figure legend: "Depletion of CDK5 decreased the cilia length in WT cells. It restored cilia length in hCDC14APD cells. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **; P≤ 0.01; **** P≤ 0.0001; ns: not significant."
NCBI gene IDs:
"hCDC14A: 8556
CDK5: 1020"
Figure legend: "CDK5 mediated cilia length decrease can be completely blocked by expression of the phospho-mimetic DBN1S142D in hCDC14APD cells. Three independent experiments; 150 cells per experiment and condition. Mean ± SD; ns = not significant; **** P≤ 0.0001."
NCBI gene IDs:
"hCDC14A: 8556
CDK5: 1020
DBN1: 1627"
Figure legend: " hCDC14APD cells showed an increased localization of myosin Va containing vesicles within the 1 μm-radius of the basal body (red cicle). The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure Quantification of C. Data was calculated from a total of 180 cells pooled from three independent experiments. Mean ± SD. * P≤ 0.05, *** P≤ 0.001, **** P≤ 0.0001 "
NCBI gene IDs:
"hCDC14A: 8556"
Figure legend: " Arp2 localization in REP1 WT, hCDC14APD and DBN1 KO cells. Signal intensity was measured 30 min after serum starvation. The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure "
NCBI gene IDs:
"hCDC14A: 8556
DBN1: 1627"
Figure legend: " Quantification of E. Sub-centrosomal (1 μm-radius; red circle in E) Arp2 was measured by quantifying the intensity of Arp2 in WT, hCDC14APD and DBN1 KO cells. Both hCDC14APD and DBN1 KO cells contain significantly higher Arp2 in the pericentrosomal area (1 μm-radius) compared to WT cells after 30 min of serum starvation. Data were calculated from a total of 120 cells pooled from three independent experiments. Mean ± SD. **** P≤ 0.0001 "
NCBI gene IDs:
"hCDC14A: 8556
DBN1: 1627"
Figure legend: " Pericentrosomal ARP2 enrichment was comparable in the RPE1 DBN1 KO cells (Tet Control) with DBN1 KO cells expressing different DBN1 versions (WT, S142A, S142D). Data were calculated from a total of 200 cells each pooled from two independent experiments. Mean ± SD. ns: not specific "
NCBI gene IDs:
"DBN1: 1627"
Figure legend: "C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL-RH6. The ratio of firefly luciferase expression to Renilla luciferase expression (expressed from co-transfected plasmid) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL-RH6. OCT4 PE-Luc ***P&lt;0.001, OCT4 DE-Luc *P&lt;0.05 (t-test). Data presented as mean ± SD (n=3)."
NCBI gene IDs:
"Luc: 
luciferase: 
OCT4: 5460"
Figure legend: " B ATAC-Seq genome tracks showing three signature peaks located near the genes SAE1, RAG1, and CD8A. Framed regions indicate cell-type-specific signature ATAC-Seq peaks. Blue triangles indicate known promoter or enhancer regions, which are annotated in the GeneHancer (GH) Regulatory Elements database "
NCBI gene IDs:
"CD8A: 925
RAG1: 5896
SAE1: 10055"
Figure legend: "A The rationale of footprinting analysis as illustrated by actual ATAC-Seq peaks, HINT-ATAC footprint hits, and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6."
NCBI gene IDs:
"RAG1: 5896"
Figure legend: "D Heatmap of VST normalized read counts (each row is also normalized by the row mean) of differentially expressed genes (pval&lt;0.1; DESeq2) in three patients in comparison to remaining 16. Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts."
NCBI gene IDs:
"DAB1: 1600
SPI1: 6688"
Figure legend: "A) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col-0 and in mkkk7. Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t=0, 10 and 30 min post induction. The p44/42 antibody was used to detect phosphorylated MAPKs. Position of the individual phosphorylated MAPKs is indicated at the right. Equal loading of proteins is shown with an α-Actin antibody as a loading control (bottom panel). Three biological replicates were done with identical results"
NCBI gene IDs:
"mkkk7: 820555"
Figure legend: "B) MPK6 phosphorylation is specifically enhanced in mkkk7. Comparison of phosphopeptide abundances from selected MAP kinases in Col-0 (blue) and mkkk7 (red) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring (SRM) mass spectrometry. Phosphopeptide corresponding to MKKK7 are only detectable in Col-0 seedlings and are non-detectable (ND) in mkkk7. Bars represent means of measured peptide areas (sum of all transition areas) for three biological replicates, with error bars ± SEM (n=3). Asterisks indicate significant difference between Col-0 and mkkk7 at individual time points (student t-test, *&gt;0.05, **&gt;0.01 and ***&gt;0.001). ND indicates integration of an area without transitions significantly above background. Above each graph the protein name and the phosphorylated residue (in brackets) is indicated as well as the corresponding phosphopeptide sequence. Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation is indicated by \"[+80]\"."
NCBI gene IDs:
"mkkk7: 820555"
Figure legend: "A) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment. Protoplasts were isolated from 4 week old plants and transfected with pWRKY29:fLUC (WRKY29) or pFRK1:fLUC(FRK1) constructs together with 35S:rLUC, as indicated in the graph. Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated. The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample, as fold induction. All measurements were normalized to the rLUC activity. Bars represent means ± STDEV (n=2). Experiment was repeated 6 times with similar results."
NCBI gene IDs:
"35S: 
LUC: 
FRK1: 816436
mkkk7: 820555
WRKY29: 828455"
Figure legend: "B) WRKY29 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 for t=0, 1, 2, and 4h. WRKY29 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p&lt;0.05, ** p&lt;0.01, student t-test)."
NCBI gene IDs:
"Ubiquitin: 
mkkk7: 820555
WRKY29: 828455"
Figure legend: "C) FRK1 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 fort=0, 1, 2, and 4h. FRK1 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p&lt;0.05, ** p&lt;0.01, student t-test)."
NCBI gene IDs:
"Ubiquitin: 
FRK1: 816436
mkkk7: 820555"
Figure legend: "A) Transient co-expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22-induced WRKY29 gene expression. Protoplasts were transfected with pWRKY29:fLUC, 35S:rLUC and indicated overexpression constructs of MKKK7 (OE-MKKK7, OE-MKKK7AA or OE-MKKK7DD) as indicated on the horizontal axis. Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs. All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample, shown as fold induction on the vertical axis. Results shown are means ± STDEV (n=2). At least two biological replicates were done with similar results"
NCBI gene IDs:
"35S: 
LUC: 
MKKK7: 820555
WRKY29: 828455"
Figure legend: "B) Quantification of bacterial growth in Arabidopsis lines Col-0, mkkk7 and p35S:MKKK7-GFP in the mkkk7 background. Four to five-week-old plants were pressure-infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re-isolated on selective media. The number of colony forming units (cfu/cm2) was determined at t=0, 2 and 3days post inoculation (dpi). Data represents mean values ± SEM (n=6; **, p&lt;0.01; paired t-test). Experiments were done at least twice with similar results."
NCBI gene IDs:
"35S: 
mkkk7: 820555
MKKK7: 820555"
Figure legend: "C) Disease symptom development in Pst-infected lines with estradiol inducible constructs of ind-MKKK7AA L8, ind-MKKK7AA L10, ind-MKKK7DD L1 and ind-MKKK7DD L3. Two independent transgenic lines for each construct were grown under short-day conditions and disease symptoms were scored 3 dpi. Data represents mean values ± SEM (n=20; *, p&lt;0.05; **, p&lt;0.01; paired t-test). The vertical axis represents the percentage disease symptoms. Experiments were done at least twice with similar results."
NCBI gene IDs:
"MKKK7: 820555"
Figure legend: "A) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col-0 and mkkk7."
NCBI gene IDs:
"mkkk7: 820555"
Figure legend: "B) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines."
NCBI gene IDs:
"MKKK7: 820555"
Figure legend: "C) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col-0 and two independent inducible MKKK7DD transgenic lines. Graphs represent means with error bars ± SEM (n=24). The vertical axis represents the relative increase in ROS production (photon counts) after PAMP treatment. At least three biological replicate experiments were done with similar results."
NCBI gene IDs:
"MKKK7: 820555"
Figure legend: "Scatter plot elucidating fold change (FC) in gene expression between samples treated with JNKi compared to vehicle (Y-axis) and expression of constitutively active JNK (MKK7-JNK) versus vector control (X-axis) from biological triplicates. Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red."
NCBI gene IDs:
"MKK7: 26400
JNK: 5599"
Figure legend: "GSEA graphs showing (C) enrichment of a mammary stem cell signature (Pece et al, 2010) in MDA231-LM2 cancer cells expressing MKK7-JNK NES, normalized enrichment score."
NCBI gene IDs:
"MKK7: 26400
JNK: 5599"
Figure legend: "GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231-LM2 cells either expressing MKK7-JNK or treated with JNKi. Mammary stem cell signatures are indicated as follows: a, up in mammary stem cells b, up in fetal mammary stem cells ; c, up in adult mammary stem cells and d, up in mammary repopulating units"
NCBI gene IDs:
"MKK7: 26400
JNK: 5599"
Figure legend: "Heat map of the JNK signature composed of 68 genes that are induced by expression of MKK7-JNK and repressed by JNKi,"
NCBI gene IDs:
"MKK7: 26400
JNK: 5599"
Figure legend: "Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK. Values are mean from 10 wells per group ± SD."
NCBI gene IDs:
"JNK: 5599"
Figure legend: "SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "SPP1 and TNC expression in MDA231-LM2 cells transduced with pLVX-puro lentivirus without a transgene (control) or expressing constitutively-active JNK (MKK7-JNK) or mutated inactive JNK (MKK7-JNK(mut))."
NCBI gene IDs:
"MKK7: 26400
JNK: 5599
SPP1: 6696
TNC: 3371"
Figure legend: "SPP1 and TNC expression in pleural effusion (1 and 2) and ascites (3 and 4) samples from four different patients treated with JNKi (4 μM, 48h) in culture. Values are mean ± SD from triplicates. The results from all four patient samples were included to calculate significance of JNKi on gene expression. SPP1, P = 0.0079; TNC, P = 0.0002."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "Analysis of SPP1 and TNC mRNA levels in SUM159-LM1 cells grown as monolayer or spheres and treated with vehicle or JNKi. For panels A-D, values represent the mean of qPCR triplicates ± SD."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "ChIP analysis of c-Jun binding to sequences in SPP1 or TNC promoters. Analysis of SPP1 and TNC promoter fragments pulled down by c-Jun and IgG antibodies. Values represent the mean of qPCR triplicates ± SD."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "Correlation analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients (GSE14020). N = 65 metastasis samples."
NCBI gene IDs:
"JUN: 3725
SPP1: 6696
TNC: 3371"
Figure legend: "Lung metastasis in Spp1+/- mice implanted with MDA231-LM2 cells expressing control shRNA (control), and Spp1-/- mice implanted with MDA231-LM2 cells independently expressing one of two different SPP1 shRNAs (SPP1-deficient). Ex vivo lung luminescence was analyzed, panel H. Boxes show the median value with upper and lower quartiles. Whiskers represent minimum and maximum values. Control n = 16 mice, SPP1 deficient (1) n = 14 mice and SPP1 deficient (2) n = 15 mice. P values were determined by a two-tailed Mann-Whitney test. (I) Representative histological images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). In histological samples, immunohistochemistry was used to determine metastatic nodules based on expression of human vimentin (arrows). Scale bar, 100 µm."
NCBI gene IDs:
"Spp1: 20750
SPP1: 20750"
Figure legend: "SUM159-LM1 cells expressing either control shRNA (control), or one of two different TNC shRNAs (shTNC 1 and 2), were injected intravenously into NSG mice. (J) Metastatic colonization was quantified by lung bioluminescence. Control and shTNC (1) n = 8 mice per group, shTNC (2) n = 7 mice. P value was determined by a two-tailed Mann-Whitney test. (K) Representative images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). Arrows denote vimentin-expressing cancer cells in metastases."
NCBI gene IDs:
"TNC: 21923"
Figure legend: "Kaplan-Meier analysis of breast cancer patients (combined GSE2603 and GSE5327 datasets) showing a link between high SPP1 and TNC expression and poor lung metastasis-free survival. Patient samples, SPP1 and TNC low n = 70; SPP1 and TNC high n = 70. HR, Hazard ratio."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours. qPCR triplicates ± SD."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi. qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P &lt; 0.05 for both vehicle and PAX-treated cells."
NCBI gene IDs:
"SPP1: 6696
TNC: 3371"
Figure legend: "Growth curves of mammary tumors in vehicle- or PAX-treated Spp1+/- mice implanted with the indicated cells expressing control shRNA, and Spp1-/- mice implanted with the indicated cells expressing SPP1 shRNA. MDA231-LM2: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 14 tumors. SUM159-LM1: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 15 tumors."
NCBI gene IDs:
"SPP1: 20750
Spp1: 20750"
Figure legend: "Quantification of lung metastasis from the animals described in panels A and B. The number of metastatic foci per field of view (FOV) or per lung section (LS) was determined. MDA231-LM2 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. SUM159-LM1 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice."
NCBI gene IDs:
"SPP1: 20750"
Figure legend: "Tumor weight in mice at day 40 after implantation of MDA231-LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin (Adriamycin) and cyclophosphamide (AC regimen). Control, AC and SPP1 def. n = 16 tumors per group, SPP1 def. + AC n = 14 tumors."
NCBI gene IDs:
"SPP1: 20750"
Figure legend: "Mammary tumor growth curves in PAX-treated NSG mice bearing control or TNC knockdown MDA231-LM2 tumors. Values are means ± SEM, n = 16 tumors per group. **** P &lt; 0.0001. Lung metastases quantified in tumor bearing mice"
NCBI gene IDs:
"TNC: 21923"
Figure legend: "(c) Pathogenic Parkin mutations abrogate mitochondrial translocation and/or clearance. HeLa cells transfected with Flag-Parkin wild-type or pathogenic mutants were treated with CCCP and stained with anti-Flag (green), anti-TOM20 (red) and Hoechst33342 (blue). Representative merged images are shown. Scale bars, 10 μm. Immunostaining of cells transfected with non-functional cysteine mutants and functional Parkin variants is shown in Supplementary Information, Fig. S1a and S1b, respectively."
NCBI gene IDs:
"Parkin: 5071"
Figure legend: "(d, e) Based on the mitochondrial effects, Parkin mutants can be classified into functional and non-functional classes. (d) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment, as defined by complete colocalization of Parkin with the mitochondrial marker (n = 3). (e) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment, as shown by complete loss of mitochondrial staining (n = 3). Data represent the mean ± s.d., *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 compared with wild-type. WT, wild-type."
NCBI gene IDs:
"Parkin: 5071"
Figure legend: "Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a-b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (- DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control."
NCBI gene IDs:
"Parkin: 5071"
Figure legend: "(c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not."
NCBI gene IDs:
"Parkin: 5071"
Figure legend: "(e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type."
NCBI gene IDs:
"Parkin: 5071"
Figure legend: "(c) Western blot of PINK1 siRNA-transfected HeLa cells used in d-h demonstrates the knockdown efficiency and specificity of PINK1 silencing."
NCBI gene IDs:
"PINK1: 65018"
Figure legend: "d-h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type and PINK1-V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm."
NCBI gene IDs:
"Parkin: 5071
PINK1: 65018"
Figure legend: "a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary Parkin of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively."
NCBI gene IDs:
"Parkin: 5071"
Figure legend: "(c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin."
NCBI gene IDs:
"ubiquitin: 6233///7311///7316"