EMBO/SourceData huggingface dataset:
Figure legend: " G Representative 3D images of ESM-1 and Caveolin-1 in pancreatic islets from Gja1i∆EC and control mice. Insets (* and #) show higher magnifications with surface rendering for ESM-1 and Caveolin-1 immunostaining. Combo plot with the quantification of ESM-1+ cell numbers per islet (f.u). (n=5), P-value, and two-tailed unpaired t-test. The scale bars for the 3D images are 50 µm, and 5 µm for the higher magnifications with single-cell resolution images. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P &lt; 0.01; ****: P &lt; 0.0001. Nuclei: TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. "
NCBI gene IDs:
"Gja1: 14609"
Figure legend: " H 3D images with CXCR4 and Emcn immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. I Representative 3D images show CXCR4, Ki67 and VE-Cadherin immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. J Combo plots show the percentage of CXCR4+ β-cells per islet and Ki67+ β-cells normalized to the total number of β-cells per islet in Gja1i∆EC mutant versus the littermate control mice. (n=5), P-values, and two-tailed unpaired t-tests. K Box whiskers, and scatter plot shows the quantification of CD31+ Emcnhi vessel density in aged Gja1i∆EC mutant and control pancreatic islets. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Combo plot (below) shows the quantification of β-cells on thick murine pancreatic sections. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Data information: The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P &lt; 0.01; ****: P &lt; 0.0001. Nuclei: TO-PRO-3"
NCBI gene IDs:
"Gja1: 14609"
Figure legend: "(B) Identification of the endogenous SUMO-sites in IRF2BP1 (left panel) and IRF2BP2 (right panel). HeLa cells were transfected with HA-tagged wildtype (WT) or mutant (KR) proteins, endogenous SUMO-IPs were performed and the HA-signal was analyzed by immunoblotting. Mutation of the C-terminal SUMO-site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells."
NCBI gene IDs:
"HA: 
IRF2BP1: 26145
IRF2BP2: 359948"
Figure legend: "(E) Stable HeLa cells expressing pIRES-empty vector, IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non-targeting (nt) siRNA. Exogenous siRNA-resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1. * refers to an unspecific band."
NCBI gene IDs:
"IRF2BP1: 26145"
Figure legend: "(F) Wt and mutant IRF2BP1 localizes in the nucleus. After knockdown of endogenous IRF2BP1, stable IRF2BP1 (WT or K579R) cell lines were immunostained for IRF2BP1. Exogenous IRF2BP1 variants show a similar nuclear localization. Scale bar = 10 µm."
NCBI gene IDs:
"IRF2BP1: 26145"
Figure legend: "(G) IRF2BP1 wt and mutant associate with chromatin to a similar extent. HeLa cells were lysed in 0.075 % NP40 (Input). After centrifugation, the nuclei were incubated and vortexed with a nuclear extract (NE) buffer containing 170 mM NaCl. The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations, first 290 mM, then 420 mM. Wildtype IRF2BP1, the SUMO-deficient K579R mutant and the SUMOylated wildtype form (*) all behave similarly."
NCBI gene IDs:
"IRF2BP1: 26145"
Figure legend: "(A) Stable IRF2BP1 cell lines (knocked-down for endogenous IRF2BP1) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour. A subset of 38 EGF-dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines (at least 1.5-fold). Among them are DUSP1 (arrow), ATF3, Fos and Egr2. Each microarray was performed in triplicates, the bars show log2 values of the fold changes for wt cells (black bars) and for KR cells (grey bars)."
NCBI gene IDs:
"ATF3: 467
DUSP1: 1843
Egr2: 1959
Fos: 2353
IRF2BP1: 26145"
Figure legend: "(B) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells. Gene architecture of human DUSP1. The primers at -243/-67 (&quot;-67&quot;), -473/-224 (&quot;-224&quot;) and -1170/-961 (&quot;-916&quot;) relative to the TSS were used for ChIP experiments. IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides -243 and -67."
NCBI gene IDs:
"DUSP1: 1843"
Figure legend: "(C) ChIP/qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between -243 and -67. Data show mean (bar) and individual data points from 2 biological replicates."
NCBI gene IDs:
"DUSP1: 1843"
Figure legend: "(D) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF. Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked-down for endogenous IRF2BP1, followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region -243 and -67. Data show means +/- SEM from 3 biological replicates."
NCBI gene IDs:
"DUSP1: 1843
IRF2BP1: 26145"
Figure legend: "(E) Q-PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1. Data show means +/- SEM from 3 biological replicates."
NCBI gene IDs:
"IR2BP1: 26145
IRF2BP1: 26145"
Figure legend: "(F) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q-PCR (left panel), data show means +/− SEM from 5 biological replicates (left panel), and IRF2BP1 protein levels. As expected, IRF2BP1 V578A is not SUMOylated (right panel). * refers to an unspecific band."
NCBI gene IDs:
"DUSP1: 1843
IRF2BP1: 26145"
Figure legend: "(G) Q-PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1. Data show means +/− SEM from 4 biological replicates."
NCBI gene IDs:
"IR2BP1: 26145
IRF2BP1: 26145"
Figure legend: "(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1. Uba2 and beta-actin serve as independent loading controls."
NCBI gene IDs:
"IRF2BP1: 26145"
Figure legend: "(C) Wound healing assay: HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days, grown to 90% confluency and analysed for wound closure with or without addition of 100 ng/ml EGF. Data show means of the relative wound density +/- SEM from 4 biological replicates."
NCBI gene IDs:
"IRF2BP1: 26145"
Figure legend: "(D) Proliferation assay: HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium. One representative biological experiment is shown with means +/-SEM from 5 technical replicates."
NCBI gene IDs:
"IRF2BP1: 26145"
Figure legend: "(E) IRF2BP1, IRF2BP2, IRF2BP1+IRF2BP2 were knocked-down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis; non-targeting siRNAs were used as a control. IRF2BP1 and IRF2BP2 have distinct and overlapping functions. Experiments were done in triplicates."
NCBI gene IDs:
"IRF2BP1: 26145
IRF2BP2: 359948"
Figure legend: "(G) Validation of two candidate genes: IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells, and cell lysates were analyzed by immunoblotting with the indicated antibodies."
NCBI gene IDs:
"IRF2BP1: 26145
IRF2BP2: 359948"
Figure legend: "(H) FACS-based analysis of EGFR surface expression: IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells, cells were serum starved for 16 hours, stained with fluorescent anti-EGFR antibodies and analyzed by flow cytometry. Shown are means of 3 independent experiments +/-SEM."
NCBI gene IDs:
"IRF2BP1: 26145
IRF2BP2: 359948"
Figure legend: "(I) Scatter plot of expression levels obtained in D. mel. S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D. mel. embryos, revealing a high correlation (Pearson coefficient 0.91) between the two datasets. Error bars represent standard deviations of 3-4 biological replicate measurements."
NCBI gene IDs:
"mNeonGreen: "
Figure legend: "(B-C) Comparison of normalized expression levels between wild-type configuration and motif knockouts for two types of core promoters (developmental: CG8157 (B); constitutive: RpL5 (C)). Upper panels: schematic depiction of the wild-type motif compositions (TTGTT motif in RpL5 is ignored due to its strong overlap with R-INR). Two-sample t-test: ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. 3-4 biological replicate measurements."
NCBI gene IDs:
"CG8157: 36700
RpL5: 3355124"
Figure legend: "(A) Intracellular endogenous Aβ42 (green), Ankyrin-G (AnkG; magenta) and GFP (blue) in siBin1- , siCD2AP- and siControl-treated primary cortical neurons (neurons) expressing GFP immunolabelled at 9 DIV with anti-Aβ42 (clone 12F4) and anti-AnkG, analysed by spinning-disk confocal microscopy. The white rectangles indicate the dendrites (Dd) and axons (Ax) magnified below showing Aβ42 in axons and dendrites outlined based on AnkG (magenta) and GFP (blue), respectively. Scale bars, 10 µm.(B) Aβ42 line profiles in dendrites (Dd) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c).(C) Aβ42 line profiles in axons (Ax) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c)."
NCBI gene IDs:
"Bin1: 30948
CD2AP: 12488"
Figure legend: "(D) Quantification of Aβ42 (12F4) intensity in cell body (CB), dendrite (Dd) and axon (Ax) (n=5, NCB=44-53, NDd=90-120, NAx=60-74; ****PCB&lt;0.0001 siBin1 vs. siControl, ***PCB&lt;0.001 siCD2AP vs. siControl, ****PDd&lt;0.0001 siBin1 vs. siControl, ****PDd&lt;0.0001 siCD2AP vs. siControl, ****PAx&lt;0.0001 siBin1 vs. siControl, t-test, mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948
CD2AP: 12488"
Figure legend: "(E) Quantification of extracellular endogenous Aβ40, Aβ42 and of Aβ42/Aβ40 ratio by ELISA analysis of conditioned media of 9 DIV siBin1, siCD2AP or siControl neurons (n=6; *PAβ40=0.0270 siBin1 vs. siControl, *PAβ42/40=0.0378 siBin1 vs. siControl, *PAβ42/40=0.0463 siCD2AP vs. siControl, t-test, mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948
CD2AP: 12488"
Figure legend: "(F) Endogenous APP and APP-CTFs levels by western blot with anti-APP antibody (Y188) of siBin1-, siCD2AP- or siControl-treated neurons at 9 DIV.(G) Quantification of APP and APP-CTFs levels normalized to APP (n=4; *PAPP=0.0355 siBin1 vs. siControl, ***PAPPCTFs/APP&lt;0.001 siBin1 vs. siControl, ****PAPPCTFs/APP&lt;0.0001 siCD2AP vs. siControl, t-test, mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948
CD2AP: 12488"
Figure legend: "N2a cells treated with siBin1, siCD2AP or siControl.(B) Endocytosed APP detected after a 10 min pulse with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(C) The amount of endocytosed APP fluorescence at 10 min was quantified and normalized to APP-RFP fluorescence (n=3, NsiControl=56, NsiBin1=70, NsiCD2AP =42; mean ± SEM).(D) Non-degraded APP detected after 10 min pulse and 60 min chase with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Cells are outlined in white. Scale bars, 10 µm.(E) APP degradation was assessed by the decrease in the amount of endocytosed APP fluorescence at 60 min relative to time 0 (10 min pulse) in siControl cells normalized to APP-RFP fluorescence (n60min=3, NsiControl=68, NsiBin1=43, NsiCD2AP=64; ****PAPP60min&lt;0.0001, t-test, mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948
CD2AP: 12488"
Figure legend: "N2a cells treated with siBin1, siCD2AP or siControl.(G) Endocytosed BACE1 detected upon 5 min pulse with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(H) The amount of endocytosed BACE1 per cell was quantified as percentage of siControl normalized to BACE1-GFP (n=3, NsiControl=86, NsiBin1=72, NsiCD2AP=113; mean ± SEM).(I) Recycled BACE1 detected at the plasma membrane of non-permeabilized cells with M1, upon a 10 min pulse, acid stripping and 20 min chase, and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(J) The amount of recycled BACE1 was quantified as in (h) (n=3, NsiControl=94, NsiBin1=58, NsiCD2AP=109; ****P&lt;0.0001 siBin1 vs. siControl, t-test, mean ± SEM).(K) Non-recycled BACE1 detected in acid-stripped permeabilized cells pulse-chased as in (i) with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(L) The amount of non-recycled BACE1 was quantified as in (h) (n=3, NsiControl=61, NsiBin1=51; ****P&lt;0.0001 siBin1 vs. siControl, t-test; n=2, NsiCD2AP=37; mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948
CD2AP: 12488"
Figure legend: "(A - D) APP endocytic trafficking followed in neurons expressing APP-RFP treated with siCD2AP or siControl using a pulse-chase assay with 22C11, analysed by epifluorescence microscopy.(A) Endocytosed APP detected with 22C11 (10 min pulse; top panels) and APP-RFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(B) The amount of endocytosed APP (10 min) in cell body, dendrites and axons normalized to APP-RFP expression in the cell body quantified as percentage of siControl (n=3, NsiControl=19, NsiCD2AP=20; mean ± SEM).(C) Endocytosed APP detected with 22C11 (10 min pulse, 60 min chase; top panels) and APP-RFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(D) The amount of endocytosed APP (60 min chase) was quantified as in (b) (n=3, NsiControl=23, NsiCD2AP=19; **PCB=0.001 siCD2AP vs. siControl, ***PDd=0.0004 siCD2AP vs. siControl, t-test; #P=0.0347 Dendrite vs. Cell body, ###P=0.0008 Dendrite vs. Axon, 1-way ANOVA with Tukey's multiple comparisons test; mean ± SEM)."
NCBI gene IDs:
"CD2AP: 12488"
Figure legend: "(E) Degradation of surface biotinylated APP (biotin APP) (time 0) chased for 20 and 60 min in neurons treated with siCD2AP, siBin1 or siControl. Biotin APP and total APP were detected with anti-APP (Y188) by western blot.(F) Quantification of biotinylated APP normalized to levels at time 0 (n=3-4, *P60min=0.0198, t-test; replicates and mean)."
NCBI gene IDs:
"Bin1: 30948
CD2AP: 12488"
Figure legend: "(A - D) BACE1 endocytic trafficking followed in siBin1- and siControl-treated neurons expressing BACE1-GFP N-terminally tagged with FLAG using a pulse/chase assay with M1, an anti-FLAG antibody, analysed by epifluorescence microscopy.(A) Endocytosed BACE1 detected with M1 (15 min pulse; top panels) and BACE1-GFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(B) The amount of endocytosed BACE1 per cell body, dendrite and axons was normalized to BACE1-GFP expression in the cell body and quantified as percentage of siControl (n=3, NsiControl=17, NsiBin1=19, mean ± SEM).(C) Recycled BACE1 detected with M1 (15 min pulse, acid stripping and 20 min chase; top panels) at the plasma membrane of non-permeabilized neurons and BACE1-GFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(D) The amount of recycled BACE1 analysed as in (b) (n=4, NsiControl=31, NsiBin1=38, ***PCB=0.0002 and ****PAx&lt;0.0001 siBin1 vs. siControl, t-test; ##P=0.0014 Axon vs. Dendrite, 1-way ANOVA with Tukey multiple comparisons test; mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948"
Figure legend: "(A) Endocytosed APP (green) detected with 22C11 (10 min pulse and 60 min chase) in EEA1-positive early endosomes (magenta) in dendrites of siCD2AP- and siControl-treated neurons expressing APP-RFP, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. The white squares indicate an EEA1-positive endosome magnified on the right. Scale bar, 1 µm.(B) Quantification of colocalisation between endocytosed APP and EEA1 in dendrites (n=3, NsiControl=26 NsiCD2AP=18; ****P&lt;0.0001, t-test; mean ± SEM)."
NCBI gene IDs:
"CD2AP: 12488"
Figure legend: "(C) Non-recycled BACE1 (green) detected with M1 (15 min pulse, acid stripping and 20 min chase) in Rab5-positive early endosomes (magenta) in axons of siControl- and siBin1-treated neurons expressing Rab5-mCherry and BACE1-GFP, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares indicate a Rab5-positive endosome magnified on the right. Scale bar, 1 µm.(D) Quantification of colocalisation between non-recycled BACE1 and Rab5 in axons (n=3, NsiControl=19, NsiBin1=24; ****P&lt;0.0001, t-test; mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948"
Figure legend: "(E) APP (magenta) and BACE1 (green) colocalisation in dendrites of siCD2AP- and siControl-treated neurons expressing APP-RFP and BACE1-GFP upon DAPT treatment, recorded by time-lapse spinning-disk confocal microscopy for 120 sec (1 fps) (see supplementary movie 1). APP and BACE1 in dendrites at 0 sec are shown and during 120 sec in kymographs (bottom panels). Dotted white lines in kymographs highlight APP vesicles positive for BACE1. Scale bar, 10 µm.(F) Quantification of the colocalisation between APP and BACE1 in dendrites (n=3, NsiControl=18, NsiCD2AP=21; ****P&lt;0.0001, t-test; mean ± SEM)"
NCBI gene IDs:
"CD2AP: 12488"
Figure legend: "(G) APP (magenta) and BACE1 (green) colocalisation in axons of siBin1- and siControl-treated neurons expressing APP-RFP and BACE1-GFP upon DAPT treatment, recorded by time-lapse spinning-disk confocal microscopy for 120 sec (1 fps) (see supplementary movie 2). APP and BACE1 in axons at 0 sec are shown and during 120 sec in kymographs (bottom panels). Dotted white lines in kymographs highlight APP vesicles positive for BACE1. Scale bar, 10 µm.(H) Quantification of the colocalisation between APP and BACE1 in axons (n=4, NsiControl =17, NsiBin1=27; ****P&lt;0.0001, t-test; mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948"
Figure legend: "(A) BACE1 carriers in axons of siBin1- or siControl-treated neurons expressing BACE1-GFP, analysed by epifluorescence microscopy. Scale bar, 10 µm. White rectangles indicate a BACE1-positive carrier magnified on the right. Scale bar, 1 µm.(B) Quantification of the percentage of siBin1- or siControl-treated neurons displaying extended BACE1 carriers (as shown in (a)); (n=3, NsiControl=67 cells, NsiBin1=68 cells; *P=0.0199, t-test, mean ± SEM).(C) Quantification of circularity of individual BACE1 carriers in axons and in dendrites of siBin1- or siControl-treated neurons. The percentage of BACE1 tubules (defined by circularity &lt; 0.5; n=2, NAx=116 and NDd=134 of siControl carriers, NAx=71 and NDd=134 of siBin1 carriers; *P=0.0105 siBin1-axons compared to siControl-axons, t-test, mean ± SEM).(D) Quantification of average size (μm2) of individual BACE1 carriers in axons and in dendrites of siBin1- or siControl-treated neurons (n=2, NAx=116 and NDd=134 siControl, NAx=71 and NDd=134 siBin1; **P=0.0019, Mann-Whitney test, mean ± SEM)."
NCBI gene IDs:
"Bin1: 30948"
Figure legend: "(E and F) BACE1 (green) exit in tubular carriers from Rab5-positive early endosomes (magenta) in axons of siBin1- (f) and siControl- (e) treated neurons expressing BACE1-GFP and Rab5-mCherry using time-lapse spinning-disk confocal microscopy (4 fps) (see EV movie 3). BACE1 and Rab5 are shown in axons at 0 sec and 4.75 sec. Scale bars, 10 µm. White rectangles indicate the region used in kymographs shown on the right. Arrows and kymographs (covering 3 or 4.75 sec) of merged BACE1 and Rab5 or BACE1 alone indicate in (E) a BACE1 punctum that exits in a tubular carrier from a Rab5-positive early endosome (asterisk) and in (F) a BACE1 tubule that emanates from a Rab5-positive early endosome. Scale bars, 1 µm"
NCBI gene IDs:
"Bin1: 30948"
Figure legend: "(A) APP detected with anti-APP (Y188, green) at early endosomes (anti-EEA1, magenta) in dendrites of siCD2AP- and siControl-treated neurons with or without DAPT treatment, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares are magnified on the right panels. Scale bar, 1 µm.(B) Quantification of colocalisation between APP and EEA1-positive dendritic endosomes (n=3, NsiControl DMSO=15, NsiControl DAPT=14; NsiCD2AP DMSO=21, NsiCD2AP DAPT=22; ****P&lt;0.0001 siCD2AP DMSO vs. siControl DMSO, ####P&lt;0.0001 siCD2AP DAPT vs. siCD2AP DMSO, t-test, mean ± SEM)."
NCBI gene IDs:
"CD2AP: 12488"
Figure legend: "(C) APP (green) distribution within enlarged Rab5QL-GFP endosomes (magenta) in dendrites of siCD2AP- or siControl-treated neurons expressing APP-RFP and Rab5QL-GFP upon DAPT treatment, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares indicate a Rab5QL-positive endosome magnified on the right. Scale bar, 1 µm.(D) Qualitative analysis of APP distribution between the lumen and the membrane of Rab5QL-endosomes (n=4, NsiControl=45 (774 endosomes), NsiCD2AP=52 (888 endosomes); *P=0.0368 siCD2AP vs. siControl, t-test, mean ± SEM)."
NCBI gene IDs:
"CD2AP: 12488"
Figure legend: "(E - F) APP (green) distribution within enlarged Rab5QL-GFP endosomes (magenta) in cell bodies of siCD2AP- (f) or siControl- (e) treated neurons expressing APP-RFP, upon DAPT treatment, analysed by spinning-disk confocal microscopy. APP (green) and Rab5QL (magenta) line intensity profiles along the endosome (see inset line) are shown on the right. Scale bars, 1 µm.(G) Quantification of the amount of APP fluorescence at the limiting endosomal membrane normalized to total APP fluorescence per endosome (n=3, NsiControl=32 endosomes (11 cells), NsiCD2AP=39 endosomes (15 cells); ****P&lt;0.0001, t-test, mean ± SEM)."
NCBI gene IDs:
"CD2AP: 12488"
Figure legend: "(H) APP detected with anti-APP (Y188, green) at early endosomes (anti-EEA1, magenta) in dendrites of siCD2AP- and siControl-treated neurons upon DAPT treatment, analysed by super-resolution dSTORM imaging. Scale bars, 200 nm."
NCBI gene IDs:
"CD2AP: 12488"
Figure legend: "(I) Qualitative analysis of super-resolved APP distribution between the lumen and the membrane of EEA1-positive endosomes of siCD2AP- or siControl-treated neurons upon DAPT treatment (n=3, NsiControl=35, NsiCD2AP=57, **P=0.0012, Mann-Whitney test, mean ± SEM)."
NCBI gene IDs:
"CD2AP: 12488"
Figure legend: "B In vitro deubiquitination of Ub chains synthesized by HOIP- and LUBEL-RBR-C. Ub chains produced by RBR-C (Human or Drosophila) with UbcH7, UbcD10 or Effete were incubated with a linear linkage-specific DUB, OTULIN (WT or catalytically dead C129A (C/A) mutant) or a Lys-linkage-specific DUB, vOTU. The DUB-treated samples were subjected to Coomassie staining or immunoblotting using anti-Ub antibody."
NCBI gene IDs:
"vOTU: 31789
OTULIN: 90268"
Figure legend: "C Thioester formation assay using Atto647-labelled Ub. Ubiquitination assay using LUBEL-RBR-C WT (left panel) or C2704A (right panel): lanes 1/2 Atto-Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub-His6. In the presence of N-terminally tagged Atto-Ub and C-terminally tagged Ub-His6, only Ub2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto-labelled Ub."
NCBI gene IDs:
"LUBEL: 33950"
Figure legend: "E Linear Ub chain formation by full-length LUBEL transient expression in insect cells. Full-length Myc-LUBEL was transiently expressed in Drosophila Schneider 2 (S2) cells, and Myc-HOIP alone or with HA-HOIL-1L in HEK293T cells. Total cell lysates (TCL) of control and transfected samples were incubated with immobilized GST-Linear-Tandem Ub binding entity (Linear-TUBE) containing three tandem repeats of ABIN-1-UBAN. Pulldown samples were blotted with anti-linear Ub antibody, while TCL were blotted with anti-Myc antibody for exogenous LUBEL and HOIP, anti-HA antibody for HOIL-1L, and anti-Tubulin antibody for loading. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band."
NCBI gene IDs:
"LUBEL: 33950
HOIL-1L: 10616
HOIP: 55072
ABIN-1: 10318"