EMBO/SourceData huggingface dataset:
Figure 1(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).(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<0.0001 siBin1 vs. siControl, ***PCB<0.001 siCD2AP vs. siControl, ****PDd<0.0001 siBin1 vs. siControl, ****PDd<0.0001 siCD2AP vs. siControl, ****PAx<0.0001 siBin1 vs. siControl, t-test, mean ± SEM).(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).(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<0.001 siBin1 vs. siControl, ****PAPPCTFs/APP<0.0001 siCD2AP vs. siControl, t-test, mean ± SEM).
Figure 2N2a 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<0.0001, t-test, mean ± SEM).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<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<0.0001 siBin1 vs. siControl, t-test; n=2, NsiCD2AP=37; mean ± SEM).
Figure 3(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).(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).(G and H) CD2AP (green) and APP (magenta) localisation in dendrites (G) and axons (I) of neurons expressing APP-RFP immunolabelled at 11 DIV with anti-CD2AP, analysed by spinning-disk confocal microscopy. Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(I) Quantification of colocalisation between CD2AP and APP in axons and dendrites (n=3-4, NDd=19, NAx=17; ****P<0.0001, t-test, mean ± SEM).(J and K) CD2AP (green) and Rab5 (magenta) localisation in dendrites (J) and axons (K) of neurons expressing Rab5-GFP analysed as in (G and H). Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(L) Quantification of colocalisation between CD2AP and Rab5-positive endosomes in dendrites (Dd) and axons (Ax) (n=3, NDd=17, NAx=23, ****P<0.0001, t-test, mean ± SEM).
Figure 4(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<0.0001 siBin1 vs. siControl, t-test; ##P=0.0014 Axon vs. Dendrite, 1-way ANOVA with Tukey multiple comparisons test; mean ± SEM).(E and F) Bin1 (green) and BACE1 (magenta) localisation in axons (E) and dendrites (F) of neurons expressing BACE1-GFP immunolabelled at 11 DIV with anti-Bin1, and analysed by spinning-disk confocal microscopy. Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(G) Quantification of colocalisation between Bin1 and BACE1 in axons and dendrites (n=3, NDd=17, NAx=21; ***P=0.0006 Axon vs. Dendrite, t-test; mean ± SEM).(H and I) Bin1 (green) and Rab5 (magenta) localisation in axons (H) and dendrites (I) of neurons expressing Rab5-GFP analysed as in (E and F). Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(J) Quantification of colocalisation between Bin1 and Rab5-positive endosomes in axons (Ax) and dendrites (Dd) (n=3, NAx=39, NDd=16, ****P<0.0001, t-test, mean ± SEM).
Figure 5(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<0.0001, t-test; mean ± SEM).(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<0.0001, t-test; mean ± SEM).(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<0.0001, t-test; mean ± SEM)(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<0.0001, t-test; mean ± SEM).
Figure 6(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 < 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).(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
Figure 7(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<0.0001 siCD2AP DMSO vs. siControl DMSO, ####P<0.0001 siCD2AP DAPT vs. siCD2AP DMSO, t-test, mean ± SEM).(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).(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<0.0001, t-test, mean ± SEM).(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.(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).(J) Quantification of the distance of APP puncta present in EEA1-positive early endosomes to its centroid over the endosome radius. The closer the ratio is to 1 the closer APP is to the endosomal membrane (i.e., distance equals radius) (n=3, NsiControl=6, NsiCD2AP =6; **P=0.0051, t-test, mean ± SEM).
Figure 1(B) Direct sequencing of PARN in a control, P1 and her parents.(D) Direct sequencing of PARN in a control, P2 and her parents.(E) Aberrant splicing products detected in P2 and her mother but not in P2's father nor in a healthy control. GAPDH RT-PCR was performed as control.(F) Detection of the exon6/7 deletion by PCR with specific primers (1F/2R).(G) PARN detection in cell lysates from P1 and P2 and a healthy control. Actin was used as loading control.(H) In vitro deadenylation activity using protein extracts from control and P1 and P2 SV40T-transformed fibroblasts. Mixing protein extracts from control together with P1 or P2 eliminated the deadenylation defect whereas pairwise mixing of P1 and P2 failed to complement the deadenylation defect.
Figure 2(A) Mean telomere length (kb) of whole blood cells from patients and their parents estimated with the TRF method.(B) Representative picture of telomeric signals used for Q-FISH analysis on metaphase spreads from SV40T-transfomed fibroblasts and Muntjac cells used to normalize the signals. (C) Individual and mean values of Q-FISH analyses for control and patients. Mean fluorescence intensity of Muntjac cells was set to 1. Error bars indicate s.d.. (D) Graphical representation of (C) showing the average of fluorescence ratios (relative to Muntjac). Error bars indicate s.e.m.. The non-parametric Kruskal-Wallis test was applied to compare relative fluorescence values between Ctl and P1, and Ctl and P2.(E) Representative pictures of nuclei from two controls and patient's primary fibroblasts showing 53BP1 foci (green) and telomeres (red, detected by Telo-FISH). Yellow arrows indicate telomere dysfunction-induced foci (TIF). The scale bar corresponds to 5µm. (F) Quantification of (E) in primary fibroblasts from two healthy controls (passages > 4) and from patient 1 (passages < 4). Control 1: n = 224; Control 2: n = 263; Patient 1: n = 231. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied when indicated.(G) Representative pictures of SA-β-galactosidase staining in control (passage 15) and P1's primary fibroblasts (passage 6). Results are expressed as the percentage of SA-β-galactosidase-positive cells (averages, lower panel). Control: n = 436; P1: n = 436. A test to compare two population proportions was applied.(H) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo-FISH. The scale bar corresponds to 1µm. (I) Quantification of terminal deletions and sister telomere losses (J) from three independent experiments (counted chromatids: Ctl1: n=2224; Ctl2: n= 5696; P1: n=4764; P2: n=7908). Averages are shown and χ2-tests were applied to compare Ctl1/2 with either P1 or P2.
Figure 3(A) Validation of PARN KO in HT1080 cells by Western blot. GAPDH is used as loading control.(B) In vitro deadenylation activity assay using protein extracts from HT1080 (WT) and HT1080PARN KO (PARN KO) cells.(C) TRAP assay using successive dilutions (500 ng, 250 ng, 125 ng and 62.5 ng) of cell extracts from WT or PARN KO cells. An internal control (IC) for PCR was used.(D) qRT-PCR analysis of hTR expression in WT and PARN KO cells. Expression levels were normalized first to ACTB and then to WT. Three independent RNA extractions were performed for each cell line. Error bars indicate s.e.m..(E) Representative pictures of FISH against hTR in WT and PARN KO cells. Arrows indicate hTR foci. Scale bar 5 μm.(F) PARN detection by Western blot in the indicated conditions. Expression was induced by incubating cells with 10 ng/ml doxycycline for 72 h. Actin is used as loading control. PARN/actin ratios, normalized to doxycycline-treated WT/PARN cells, are shown below.(G) In vitro deadenylation activity assay using protein extracts from doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells.(H) qRT-PCR analysis of the indicated gene transcripts in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to WT/empty. Three independent doxycycline inductions were performed for each cell line. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.(I) Representative western blot analysis of PARN, TRF2 and p53. Actin was used as loading control. Mean TRF2/Actin levels from two independent experiments are indicated below.(J) Same as (H) for p53 and p21 transcripts. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.(K) qRT-PCR analysis of TERRA from the indicated chromosome ends in doxycycline-treated KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to KO/empty. Three independent doxycycline inductions were performed for each cell line. Error bars indicate s.e.m..
Figure 4(A) Analysis of telomeric aberrations by Telo-FISH in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Two independent experiments were performed. Counted chromatids: WT/empty: n=1824; WT/PARN: n= 1744; KO/empty: n=1452; KO/PARN: n=1920. Averages are shown and χ2-tests were applied to compare KO/empty with either WT/empty or KO/PARN.(B) TRF analysis of telomere length (kb) in KO/empty and KO/PARN cells treated with 10 ng/ml Dox for 72h. A control Western blot with PARN and Actin is shown below for the corresponding samples.
Figure 5(A) HT1080 (WT) and HT1080PARN KO (KO) cells stably transfected with GFP plasmid (empty) were transfected with siRNAs against p53 for 72h before RNA extraction and qRT-PCR. siRNAs against luciferase (siLuci) were used as control. p53 expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m.(B) Cells were treated as in (A) and transcript levels of the indicated genes were measured by qRT-PCR. cDNA expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m.(C) qRT-PCR analyses of the indicated genes were performed on RNA extracted from P1's primary or SV40T-transformed fibroblasts. cDNA expression levels were normalized to primary fibroblasts.
Figure 6(C) Northern blot analysis of pre-rRNAs from control and patient B-LCLs. (D) Log2 values of 18S-E/21S and 18S-EFL/21S for P1 and P2 were normalized to the values of the control and presented in a graphical format, as Log averages + standard deviation for two independent experiments.(E) Northern blot analysis of pre-rRNAs from HT1080PARN WT and HT1080PARN KO cells, transduced with an empty vector. (F) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector. Western blot relative to actin assessed PARN levels. (G) Quantitative analyses of 18S-E/21S and 18S-E FL/21S ratios were as described in (D) for HT1080PARN KO cells relative to HT1080PARN WT (left panel; see (E)), and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector (right panel; see (F)), with Log averages + standard deviation for four independent experiments.
Figure 7(A) Western blot analysis of Parn expression in control and Parn+/- MEFs.(B) Northern blot analysis of total RNAs from WT and Parn+/- MEFs.(D) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analyzed. Statistical significance (P = 1.5417E-08) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (E) Frequencies of expected and observed E11.5 embryos of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analysed. Statistical significance (P = 0.02535) of the differences between the observed and expected genotype distributions was assessed by χ2-test.(F), (G) p53 knock-out does not rescue Parn-/-. No Parn-/- p53-/- mouse was born from various combinations of Parn+/- and either p53+/- or p53-/- crosses (49 and 56 pups, respectively). Statistical significance (P = 0.000886) of the differences between the observed and expected genotype distributions was assessed by χ2-test.
Figure 1(C) Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp-In/T-REx HeLa cells.(D) APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n=2 independent experiments. Scale bars: 10µm or 5µm in enlarged views.
Figure 2(C) ProHits-viz generated dotblots of RAB effectors, GEFs, GAPs and sorting complexes. Green, blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins.