Journal: Neural Regeneration Research

Article Title: Fidgetin interacting with microtubule end binding protein EB3 affects axonal regrowth in spinal cord injury

doi: 10.4103/1673-5374.373716

Figure Lengend Snippet: Depletion of Fign improves functional recovery by increasing Tyr-tubulin. (A, B) Western blotting of tyrosinated (Tyr)-tubulin and Fign after spinal cord contusion (A) and sciatic nerve crush (B). α/β-Tubulin was used as a loading control. The data are shown as mean ± SE ( n = 3). The data were normalized by control (0 day) group. Linear regression showed expression trends of target proteins after injury. (C, D) Model of Fign depletion in spinal cord injury: U6-GFP Fign-AAV9 virus injected at T8, spinal cord contusion at T10 (C). Timeline of virus injection, spinal cord contusion, and Basso-Beattie-Bresnahan (BBB) evaluation (D). (E) BBB scores after spinal cord contusion (mean ± SE, n = 12 or 13). * P < 0.05; # P < 0.05, vs . Ctrl shRNA group (two-way analysis of variance followed by post hoc Bonferroni’s test). (F) Representative western blotting of Fign from injured spinal cord at 14 days after spinal cord contusion. α/β-Tubulin was used as a loading control (mean ± SE, n = 4). The data were normalized by Ctrl shRNA group. The data were analyzed by Student’s t -test. (G) Representative immunostaining results of NF200 (red, Cy3) in the spinal cord lesion zone at 14 days after spinal cord contusion and treatment with Ctrl or Fign shRNA. Immunopositivity of NF200 was markedly increased in the injured area in the Fign shRNA group. Scale bars: 500 μm, 100 μm (enlarged images). (H) Statistical results of relative NF200 levels (mean ± SE, n = 8, Student’s t -test). (I) Representative immunostaining results of Tyr-tubulin and EB3 in the spinal cord lesion zone at 14 days after spinal cord contusion and treatment with Ctrl or Fign shRNA. Microtubule plus-end binding protein EB3 significantly increased and mostly concentrated in Tyr-tubulin enriched areas in the Fign shRNA group. Tyr-tubulin is labeled in red (Alexa Fluor 647) and EB3 in yellow (Cy3) (changed to green by pseudo-color). Scale bars: 500 μm, 100 μm (enlarged images). (J) Statistical results of relative Tyr-tubulin and EB3 levels (mean ± SE, n = 5). The data were normalized by Ctrl shRNA group. The data were analyzed by Student’s t -test. AAV: Adeno-associated virus; BBB: Basso-Beattie-Bresnahan; EB3: end binding protein 3; Fign: fidgetin; GFP: green fluorescent protein; NF200: neurofilament-200; SCI: spinal cord injury; shRNA: short hairpin RNA; Tyr-tubulin: tyrosinated-tubulin.

Article Snippet: After transfer to a polyvinylidene fluoride membrane (Millipore, Boston, MA, USA), the membrane was blocked and then incubated overnight with primary antibodies at 4°C: anti-α/β tubulin (rabbit, 1:2000, CST, Danvers, MA, USA, Cat# 2148, RRID: AB_2288042), anti-tyrosinated tubulin (rat, 1:800, Abcam, Cambridge, UK, Cat# ab6160, RRID: AB_305328), anti-acetylated tubulin (mouse, 1:800, Abcam, Cat# ab24610, RRID: AB_448182), anti-Fign (mouse, 1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-514956), anti-GFP (rabbit, 1:1000, CST, Cat# 2555, RRID: AB_10692764), and anti-EB3 (rabbit, 1:20,000, Abcam, Cat# ab157217, RRID: AB_2890656).

Techniques: Functional Assay, Western Blot, Expressing, Injection, shRNA, Immunostaining, Binding Assay, Labeling

Journal: Neural Regeneration Research

Article Title: Fidgetin interacting with microtubule end binding protein EB3 affects axonal regrowth in spinal cord injury

doi: 10.4103/1673-5374.373716

Figure Lengend Snippet: Fign regulates Tyr-tubulin by binding to EB3 and parking at microtubules plus-ends. (A) Representative inverted immunostaining of Fign (green, Alexa Fluor 488), tyrosinated (Tyr)-tubulin (red, Cy3), and actin (violet, Alexa Fluor 647). Scale bars: 20 μm, 5 μm (enlarged images). (B) Representative western blotting with GFP antibody after transfection of GFP or GFP-Fign plasmid to Neuro-2a cells for 2 days. (C) Representative western blotting of tubulin proteins after transfection of GFP or GFP-Fign plasmid to Neuro-2a cells for 2 days. The data (normalized by control GFP plasmid transfection) are shown as mean ± SE ( n = 3), and were analyzed by Student’s t -test. (D) Representative inverted immunostaining of Tyr-tubulin (red, Cy3) and acetylated-tubulin (green, Alexa Fluor 488) after E18 cortical neurons were transfected with Ctrl or EB3 siRNA and lentivirus (LV)5-NC or LV5-Fign. Ctrl siRNA combined with LV5-Fign decreased Tyr-tubulin and axonal length. EB3 siRNA combined with LV5-Fign rescued this phenomenon. Scale bar: 20 μm. (E) Statistical results of relative Tyr-tubulin levels (left panel, n = 15, normalized by Ctrl siRNA + LV5-NC group) and axonal length (right panel, n = 35) of neurons transfected with Ctrl or EB3 siRNA, together with LV5-NC or LV5-Fign. The data are shown as mean ± SE and were analyzed using one-way analysis of variance and Dunnett’s test. (F) Representative inverted immunostaining of Fign in E18 cortical neurons transfected with LV5-NC or LV5-Fign. Scale bar: 20 μm. Red dotted box shows Fign in the axon. Red arrow shows the axonal terminal. Ace-tubulin: Acetylated-tubulin; E18: rat embryo at 18 days; EB3: end binding protein 3; Fign: fidgetin; GFP: green fluorescent protein; LV: lentivirus; P: pellet, tubulins in polymerized microtubules; S: supernatant, soluble tubulins; siRNA: small interfering RNA; T: total, total tubulins; Tyr-tubulin: tyrosinated-tubulin.

Article Snippet: After transfer to a polyvinylidene fluoride membrane (Millipore, Boston, MA, USA), the membrane was blocked and then incubated overnight with primary antibodies at 4°C: anti-α/β tubulin (rabbit, 1:2000, CST, Danvers, MA, USA, Cat# 2148, RRID: AB_2288042), anti-tyrosinated tubulin (rat, 1:800, Abcam, Cambridge, UK, Cat# ab6160, RRID: AB_305328), anti-acetylated tubulin (mouse, 1:800, Abcam, Cat# ab24610, RRID: AB_448182), anti-Fign (mouse, 1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-514956), anti-GFP (rabbit, 1:1000, CST, Cat# 2555, RRID: AB_10692764), and anti-EB3 (rabbit, 1:20,000, Abcam, Cat# ab157217, RRID: AB_2890656).

Techniques: Binding Assay, Immunostaining, Western Blot, Transfection, Plasmid Preparation, Small Interfering RNA

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a-b, KPC1 PDAC (a) or KP1 LUAD (b) tumor cells expressing luciferase-GFP were injected i.v. or orthotopically into the pancreas of 8-12 week old C57BL/6 female mice. Following tumor formation in the lungs or pancreas, mice were treated with vehicle (V) or combined trametinib (1mg/kg body weight) and palbociclib (100 mg/kg body weight) (T/P) for 2 weeks (left). Right, flow cytometry analysis of NK cell numbers and degranulation in each condition (a) NK cell numbers PIP V, PIP TP and PIL TP, n=10; PIL V, n=9) and degranulation (n=5 independent mice per group). (b) NK cell numbers LIL V, n=3: LIL TP, n=5; LIP V, n=13; LIP TP, n=15 and degranulation LIP V, n=5; LIP TP, n=7 independent mice). Data represents pool of 3 independent experiments. c, KPC1 PDAC or KP1 LUAD cells expressing luciferase-GFP were injected orthotopically into the livers of 8-12 week old C57BL/6 female mice and treated as in (a) following tumor formation (left). Right, flow cytometry analysis of NK cell numbers Data represents pool of 2 independent experiments. (c) NK cell numbers and NK degranulation PILiver V, n= 9; PILiver TP, n=10 and LILiver V, n=8; LILiver TP, n=10 independent mice). d, Kaplan-Meier survival curve of C57BL/6 mice harboring KPC1 PDAC tumors in pancreas (PIP) treated with vehicle or trametinib (1 mg/kg) and palbociclib (100 mg/kg) in the presence or absence of a NK1.1 depleting antibody (PK136; 250 ug) (V, n=5; TP and TP+αNK1.1, n=7 independent mice). e, Kaplan-Meier survival curve of C57BL/6 mice harboring KPC1 PDAC tumors in lungs (PIL) and treated as in (d) (V, n=10; TP, n=9 and TP+αNK1.1, n=8 independent mice). f, Kaplan-Meier survival curve of C57BL/6 mice harboring KP2 LUAD tumors in the lungs (LIL) and treated as in (d) (V, n=6; TP, n=7 and TP+αNK1.1, n=8 independent mice). g, Kaplan-Meier survival curve of C57BL/6 mice harboring KP1 LUAD tumors in pancreas (LIP) and treated as in (d) (V, n=7; TP and TP+αNK1.1, n=8 independent mice). P values in a-c were calculated using two-tailed, unpaired Student’s t-test, and those in d-g calculated using log-rank test. Error bars, mean ± SEM.

Article Snippet: The following primary antibodies were used: H3K27me3 (9733; 1:400) and GFP (2956; 1:200) (Cell Signaling); p21 (556431; 1:200) (BD Biosciences); and GFP (AB6673; 1:250) (Abcam).

Techniques: Expressing, Luciferase, Injection, Flow Cytometry, Two Tailed Test

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a, Representative Haematoxylin and eosin (H&E) (top) and Masson’s trichrome (bottom) staining of indicated KPC1 PDAC (PIP, PIL, PILiver) and KP1 LUAD (LIL, LIP, LILiver) derived-tumors from 2-3 independent experiments. Scale bars, 100μm. b, Immunohistochemical (IHC) staining of indicated KPC1 PDAC (PIP, PIL, PILiver) and KP1 LUAD (LIL, LIP, LILiver) derived-tumors treated with vehicle (V) or combined trametinib (1mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. Quantification of the percentage of SA-β-gal+ area and the number of Ki67+ and pRb+ cells per field are shown inset (n=2-4 per group). Scale bar, 50μm. c, Immunofluorescence staining of indicated KPC1 PDAC (PIP, PIL, PILiver) and KP1 LUAD (LIL, LIP, LILiver) derived-tumors grown in different organs and treated as in (b). Quantification of the percentage of GFP+ (green) tumor cells expressing p21 (cyan) is shown inset (n=2-4 per group). Scale bar, 50μm. d, GFP+ tumor cells were FACS sorted from indicated tumors and extracted RNA subjected to RNA-seq analysis (n=2-4 per group). Gene Set Enrichment Analysis (GSEA) of RNA-seq data using an established senescence gene set is shown. NES, normalized enrichment score. P values in d were calculated using two-sided, Kolmogorov-Smirnov test. Error bars, mean ± SEM.

Article Snippet: The following primary antibodies were used: H3K27me3 (9733; 1:400) and GFP (2956; 1:200) (Cell Signaling); p21 (556431; 1:200) (BD Biosciences); and GFP (AB6673; 1:250) (Abcam).

Techniques: In Vivo, Staining, Derivative Assay, Immunohistochemistry, Immunofluorescence, Expressing, RNA Sequencing Assay

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a, KPC1 PDAC or KP1 LUAD tumor cells expressing luciferase-GFP were injected i.v. or orthotopically into the pancreas of 8-12 week old C57BL/6 female mice. Following tumor formation in the lungs or pancreas, mice were treated with vehicle (V) or combined trametinib (1mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. GFP+ tumor cells were FACS sorted and extracted RNA subjected to RNA-seq analysis (n=2-4 per group). b, KEGG pathway analysis of pathways enriched in tumors in the lungs (LIL, PIL) compared to tumors in the pancreas (PIP, LIP) following T/P treatment. c, Heatmap showing fold change in SASP gene expression following T/P treatment in indicated tumor settings. d, Fold change in expression of select SASP chemokines following T/P treatment in indicated tumor settings (n=2-4 per group). Error bars, mean ± SEM. e, Transcription factor enrichment analysis showing transcriptional regulators whose targets are differentially expressed in tumors in the pancreas (PIP, LIP) following T/P treatment. f, Gene Set Enrichment Analysis (GSEA) of EZH2 transcriptional targets. NES, normalized enrichment score. g, Immunofluorescence staining of indicated KPC1 PDAC (PIP, PIL) and KP1 LUAD (LIL, LIP) derived-tumors grown in different organs and treated with vehicle (V) or combined trametinib (1mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. Quantification of the percentage GFP+ (green) tumor cells expressing H3K27me3 (cyan) is shown inset (PIP V; PIP TP; PIL TP; LIL TP and LIP TP, n=3, PIL V; LIL V and LIP V, n=2 independent tumors). Scale bar, 50μm. P values in b were calculated using two-sided, hypergeometric test and those in f using two-sided, Kolmogorov-Smirnov test.

Article Snippet: The following primary antibodies were used: H3K27me3 (9733; 1:400) and GFP (2956; 1:200) (Cell Signaling); p21 (556431; 1:200) (BD Biosciences); and GFP (AB6673; 1:250) (Abcam).

Techniques: Expressing, Luciferase, Injection, RNA Sequencing Assay, Immunofluorescence, Staining, Derivative Assay

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a, Schematic of KPC PDAC syngeneic orthotopic transplantation into 8-16 week old male and female SMA-TK mice and treatment regimens. b, Immunohistochemical (IHC) staining of KPC1 orthotopic PDAC tumors propagated in SMA-TK mice treated with vehicle, trametinib (1 mg/kg) and palbociclib (100 mg/kg), and/or ganciclovir (GCV) (50 mg/kg) for 2 weeks. Quantification of number of SMA+ cells per field is shown inset (b, V, n=5; TP, n=4; GCV, n=4; and TP/GCV, n=5 independent tumors). Scale bar, 50μm. c, Waterfall plot of the response of KPC1 orthotopic PDAC tumors propagated in SMA-TK mice to treatment as in (b) (c, V, n=6; TP, n=10; GCV, n=8, and TP/GCV, n=15 independent mice). Data represents pool of 2 independent experiments. d-e, Flow cytometry analysis of NK (d) and T cell (e) numbers and activation markers in KPC1 orthotopic PDAC tumors propagated in SMA-TK mice following treatment as in (b) (n=8-16 per group). (d-e, V, n=8; TP, n=9; GCV, n=8, and TP/GCV, n=16 independent mice). Data represents pool of 2 independent experiments. Error bars, mean ± SEM. f, KEGG (left) and REACTOME (right) pathway analysis of RNA-seq data generated from FACS sorted GFP+ tumor cells from SMA-TK mice harboring KPC1 orthotopic PDAC tumors and treated as in (b) (n=4-5 per group). g, Heatmap of RNA-seq analysis of SASP gene expression in PDAC cells from KPC1 orthotopic PDAC tumors propagated in SMA-TK mice and treated as in (b) (n=4-5 per group). h, Transcription factor enrichment analysis showing transcriptional regulators whose targets are differentially expressed in tumor cells from KPC1 orthotopic PDAC propagated in SMA-TK mice treated with T/P alone compared with combined T/P/GCV treatment. i, Gene Set Enrichment Analysis (GSEA) of EZH2 transcriptional targets. NES, normalized enrichment score. j, Immunofluorescence staining of KPC1 orthotopic PDAC tumors propagated in SMA-TK mice treated as in (b). Quantification of the percentage GFP+ (green) tumor cells expressing H3K27me3 (cyan) is shown inset (n=2 per group). Scale bar, 50μm. P values in c-e were calculated using two-tailed, unpaired Student’s t-test, f using two-sided, hypergeometric test, and i using two-sided, Kolmogorov-Smirnov test.

Article Snippet: The following primary antibodies were used: H3K27me3 (9733; 1:400) and GFP (2956; 1:200) (Cell Signaling); p21 (556431; 1:200) (BD Biosciences); and GFP (AB6673; 1:250) (Abcam).

Techniques: Transplantation Assay, Immunohistochemistry, Flow Cytometry, Activation Assay, RNA Sequencing Assay, Generated, Expressing, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature cancer

Article Title: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

doi: 10.1038/s43018-023-00553-8

Figure Lengend Snippet: a-b, KPC2 PDAC or KP2 LUAD tumor cells expressing GFP were injected i.v. or orthotopically into the pancreas of 8-12 week old C57BL/6 female mice. Following tumor formation, mice were treated with vehicle (V) or combined trametinib (1mg/kg) and palbociclib (100 mg/kg) (T/P) for 2 weeks. Flow cytometry analysis of NK cell numbers and degranulation in PDAC (PIP, PIL) (a) and LUAD-derived tumors (LIL, LIP) (b) grown in different organs are shown (a-b, PIP V, n=3; PIP TP, n=4; PIL V and PIL TP, n=8 independent mice). c, Flow cytometry analysis of NK cell numbers and degranulation in spleens of mice with KPC1-derived PIP tumors treated as in (a) (n=5 independent mice per group). d, Kaplan-Meier survival curve of mice with KPC2-derived PIP tumors treated with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or depleting antibodies against NK1.1 (PK136; 250 μg) or CD8 (2.43; 200 μg) (d, V, n=5; TP; TP+αNK1.1 and TP+αCD8, n=8). e, IVIS images showing luciferase signaling in KPC1-derived PIL tumors following treatment as in (a). Right, quantification of total luminescence in the thoracic region (e, V, n=5; TP and TP+αNK1.1, n=8 independent mice). f, Waterfall plot of the response of KPC1-derived PIP tumors following 2 week treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or an NK1.1 depleting antibody (PK136; 250 μg) (f, V and TP, n=5, TP+αNK1.1, n=6 independent mice). g, Waterfall plot of the response of KPC2-derived PIP tumors following 2 week treatment with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or an NK1.1 (PK136; 250 μg) or CD8 (2.43; 200 μg) depleting antibody (g, V, n=5; TP, n=7; TP+αNK1.1 and TP+αCD8 ,n=8 independent mice). h, Kaplan-Meier survival curve of mice with KPC1-derived PIL tumors treated with vehicle, combined trametinib (1mg/kg) and palbociclib (100mg/kg), and/or depleting antibodies against CD8 (2.43; 200 μg) or CD4 (GK1.5; 200 μg) (h, V, n=5; TP, TP+αNK1.1 and TP+αCD8, n=7 independent mice). i, Flow cytometry analysis of CD4+ and CD8+ T cell numbers and degranulation in KPC1 PDAC (PIP, PIL, PILiver) and KP1 LUAD-derived tumors (LIL, LIP, LILiver) grown in different organs and treated as in (a) (i, PIP V, PIP TP, PIL TP, n=10; PIL V, n=9; LIL V, n=3; LIL TP, n=5; LIP V, n=13; LIP TP, n=15; PILiver V, n=9; PILiver TP, n=10; LILiver V, n=8; LILiver TP, n=10 independent mice). Data represents pool of 3 independent experiments. P values in a-c, e-g, and i were calculated using two-tailed, unpaired Student’s t-test, and those in d and h were calculated using log-rank test. Error bars, mean ± SEM.

Article Snippet: The following primary antibodies were used: H3K27me3 (9733; 1:400) and GFP (2956; 1:200) (Cell Signaling); p21 (556431; 1:200) (BD Biosciences); and GFP (AB6673; 1:250) (Abcam).

Techniques: Expressing, Injection, Flow Cytometry, Derivative Assay, Luciferase, Two Tailed Test

Journal: bioRxiv

Article Title: Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species

doi: 10.1101/2023.09.20.558718

Figure Lengend Snippet: (A) Accessibility profiles of astrocyte-specific enhancers in the human whole-body accessibility atlas . Single-cell profiles were grouped within each tissue into pseudo-bulk aggregates, then normalized according to the signal (reads in peaks) within the dataset. Accessibility profiles are likely to predict enhancer activities within each tissue. Focusing on liver, some astrocyte-specific enhancers are predicted to have high expression, and some are predicted to have very little or no expression. In contrast, accessibility atlases do not predict expression of GFAP promoter across tissues. (B) Whole livers from mice injected intravenously with eHGT_381h- and eHGT_390m-enhancer-AAV vectors, stained with anti-GFP antibody. eHGT_381h has high liver accessibility, is predicted to have high liver expression, and shows many strong SYFP2-expressing hepatocytes throughout the liver as predicted. In contrast, eHGT_390m has very little liver accessibility and so is predicted to have little liver expression, and in fact shows few positive SYFP2-expressing hepatocytes as predicted. (C) Agreement between liver expression predictions and liver expression measurements across several astrocyte-specific enhancer-AAV vectors. eHGT_371m, 371h, 381h, and 386m all show many SYFP2-expressing hepatocytes as predicted. eHGT_390m, 390h, 375m, and 387m show few weak SYFP2-expressing hepatocytes as predicted. GFAP promoter shows many expressing hepatocytes, which was not predictable from the accessibility atlases. eHGT_380h shows many SYFP2-expressing astrocytes, in contrast to the epigenetic prediction. Liver images in B and C represent one to two mice analyzed for each vector. (D-E) Testing fidelity of enhancer-AAV expression across disease states. We used a Dravet syndrome model Scn1a R613X/+ mouse to induce epilepsy-associated hippocampal gliosis, injected enhancer-AAVs prior to the critical period, and analyzed tissue for expression patterns after the critical period (D). We assessed hippocampal gliosis with anti-GFAP antibody and enhancer-AAV expression with anti-GFP antibody (E). eHGT_390m maintained specific expression and similar levels in hippocampal astrocytes regardless of epileptic gliosis. In contrast, GFAP promoter expression strongly increased in gliotic astrocytes, and also was observed in dentate gyrus granule cells. Red dashed rectangles indicate the position of the expanded zoomed view, and the curved arrows indicate a rotated view. Abbreviations: ML molecular layer, GCL granule cell layer, PoL polymorphic layer.

Article Snippet: For IHC we used the following antibodies: chicken anti-GFP (Aves # GFP-1010), rabbit anti-Sox9 (Cell Signaling clone D8G8H, # 82630S), mouse CC1 antibody (Abcam # ab16794), mouse anti-GFAP (Millipore Sigma clone G-A-5, # G3893), with 5% normal goat serum (Thermo Fisher Scientific # 31872) and 0.1% Triton X-100 (VWR 97062-208) for blocking and permeabilization, and appropriate Alexa Fluor-conjugated secondary antibodies for detection.

Techniques: Expressing, Injection, Staining, Plasmid Preparation

Journal: bioRxiv

Article Title: Proteome-scale characterisation of protein motif interactome rewiring by disease mutations

doi: 10.1101/2023.09.18.558189

Figure Lengend Snippet: A. Left: Consensus motif of KEAP1 KELCH as established with the HD2 library and the aligned SQSTM1 peptide with the P348L mutation site indicated in blue. Right: Displacement curves from KEAP1 KELCH domain with wild-type/P348L SQSTM1 334-349 peptide. Measurements were in at least technical triplets. B. GFP trap of YFP or GFPKEAP1 and probing for co-immunoprecipitation of wild-type/P348L Myc-SQSTM1 in HeLa cells (n=3). C-D . Network of KEAP1 ( C ) or KPNA4 ( D ) with the prey proteins from the domain-mutation pairs. Interactions and corresponding mutations disrupted by the mutation are indicated in blue, enhanced in red and neutral effects in grey. A green dot indicates previously reported interacting prey proteins and a straight line if the domain’s binding motif is found in the binding peptide. The circle size encodes the confidence level of the domain-mutation pairs. For KPNA4 the network has been reduced to include the reported interactors and the domain-mutation pairs with an associated p-value ≤ 0.001. E. Left: Consensus motif of KPNA4 ARM as established with the HD2 library and the aligned CDC45 and ABRAXAS1 peptides with the mutation sites indicated in blue. Right: Displacement curves from KPNA4 ARM domain with wild-type/R157C CDC45 152-166 and wild-type/R361Q ABRAXAS1 349-364 peptides. Measurements were in technical triplets. F-G. Representative images of the localisation of wild-type and mutant EGFP-tagged (R361Q) ABRAXAS1 and (R157C) CDC45 in relation to the nuclear Hoechst staining (n=3). H. GFP trap of EGFP (n=3), wildtype EGFP-CDC45 (n=3) or mutant R157C EGFP-CDC45 (n=2) and probing for co-immunoprecipitation of T7-KPNA7 in HEK293T cells.

Article Snippet: For visualisation, mouse anti-Myc antibody (1:1000, Thermo Scientific), mouse anti-Flag antibody (1:5000, Sigma Aldrich), rabbit anti-T7 (1:1000, Cell signalling), rabbit anti-GFP antibody (1:5000 in house) and mouse anti-GFP (1:1000, Roche) antibody were used and the membranes incubated overnight at 4 °C with the antibodies in 2.5 % milk in 1xPBS with 0.1% Tween-20.

Techniques: Mutagenesis, Immunoprecipitation, Binding Assay, Staining