EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. Diagram of Sre2MS substrate (aa 423-793). The enlarged model shows predicted positions of TM1 and TM2 denoting the 26 amino acids tested for mutagenesis by colored squares. Numbers indicate the number of amino acid residues. Mutants showing a block in cleavage due to defects in Dsc1 function are indicated in orange, Rbd2 function in pink, a combination of defects in Dsc1 and Rbd2 in blue (intermediate), and residues showing normal cleavage are indicated in gray. B. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. C. Model outlining the predicted fate of Sre2MS in different yeast strains. U denotes either mono-ubiquitin or poly-ubiquitin. D-E. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. Asterisks indicate non-specific, loading control bands, which were used to normalize the intensities of P and N in Figure 5F. Two independent isolates, A and B, are shown for each strain. F. Intensities of individual P and N bands from western blots were quantified using LI-COR software. Relative intensity of P and N to corresponding non-specific band (asterisk) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis. The ratio of N to P or relative P level was presented in bar graphs. G. The average ratio of N to P level for different Sre2MS mutants in wild-type cells (Appendix Fig S2) was plotted. Cleavage was scored as normal if the ratio was >2. H. For Sre2MS mutants showing defects in cleavage, the average fold-increase in P level in dsc1Δ cells compared to WT cells from two isolates (Appendix Fig S2) was plotted. Mutants were grouped into three phenotypic classes (intermediate, dsc1Δ, and rbd2∆) according to observed precursor accumulation. Precursor accumulation was defined as dsc1Δ if <1.5 (orange), rbd2Δ if >3 (pink), and intermediate if in between (blue).
Figure 2Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet, and activated by anti-CD3ε/CD28 Dynabeads for 2 days, in the presence of different [EGTA] in the medium. CellTrace Violet/CTV signal in living cells at the end of the culture is shown. Cell proliferation was quantified as division index based on CTV signals in (a). EGTA was added 48h after the start of culture (day 2), and the percentage of iNKT cells Thymic lobes from new born (day 0) BALB/c mice were cultured in vitro for 6 days. the percentage of Tbethi population in iNKT cells Thymic lobes from new born (day 0) BALB/c mice were cultured in vitro for 6 days. EGTA was added 48h after the start of culture (day 2), and the expression of CXCR3 (g), PLZF (h) and PD1 (i) in total iNKT cells are shown.
Mitotic chromosome proteins in MKI67 KO HeLa cells.Figure 7A-B. GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells. A. GNL3 (HPA036743). Median overlapKO = 0.539, median overlapWT = 0.795 (P = 9.34x10-9) and median Pearson correlationKO = -0.335, median Pearson correlationWT = 0.134 (P = 3.58x10-6). B. PES1 (HPA040210). Median overlapKO = 0.516, median overlapWT = 0.755 (P = 1.77x10-13) and median Pearson correlationKO = -0.293, median Pearson correlationWT = 0.129 (P = 4.06x10-13). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. C. No protein aggregates were observed in MKI67 KO cells for PWP1 (HPA038708), despite being localized to mitotic chromosomes in WT cells. Median overlapKO = 0.523, median overlapWT = 0.861 (P = 1.05x10-13) and median Pearson correlationKO = -0.190, median Pearson correlationWT = 0.515 (P = 8.71x10-15). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median. D-E. Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells. White arrows indicate where protein aggregates can be seen. D. DENND4A (HPA065343). Median overlapKO = 0.671, median overlapWT = 0.803 (P = 1.02x10-4) and median Pearson correlationKO = -0.224, median Pearson correlationWT = 0.236 (P = 1.91x10-5). E. NOL12 (HPA003547). Median overlapKO = 0.468, median overlapWT = 0.820 (P = 1.01x10-17) and median Pearson correlationKO = -0.318, median Pearson correlationWT = 0.381 (P = 1.81x10-10). White arrow indicates metaphase cell. Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.
24F4A mediates BDCA2 internalization and type I IFN inhibition in vivo.Figure 3Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A-C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3). Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.D PK/PD relationship between 24F4Aserum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i-iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested. Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).
SATB2 interacts with the INM protein LEMD2 Model of a hypothetical NL-chromatin tether containing SATB2.Figure 1Model of a hypothetical NL-chromatin tether containing SATB2. Schematic representation of the nuclear envelope consisting of two lipid bilayers, the inner nuclear membrane (INM) and outer nuclear membrane (ONM), and the lamin polymer underlying the INM (modified after Zuleger et al., 2011). Depicted are the INM protein LEMD2, lamins and BAF, identified as SATB2 interaction partners (Cera et al., 2019). Immunoprecipitation of SATB2 from mouse neonatal cortical lysates. LEMD2 was detected by Western blotting in the SATB2-immunoprecipitate from control but not SATB2-deficient cortical lysate. The equal input of total protein from control and SATB2-deficient cortical lysates was controlled by ERK2 detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads). Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody, followed by WB detection of LEMD2 and SATB2. Lysates from primary cortical culture lysates were used. GST pull-down assays. Representative images of n = 3 independent experiments are shown. GST pull-down assay of transiently overexpressed V5-tagged LEMD2 (i), SATB2 (ii), and LEMD2ΔLEM (iii) from HeLa cell lysates using GST, GST-SATB2, and GST-LEMD2(413-503) hybrid proteins. Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5-tagged full-length SATB2 (Satb21-733) and EGFP as control. The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker). The CUT-like domain of SATB2 is required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with V5-tagged Satb21-247 and Satb21-157 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT-like domain (Satb21-247). The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker). CUT1 and CUT2 domains are required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from HeLa cells transfected with V5-tagged Satb2346-733 and Satb2616-733 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant (Satb2346-733).
Peripheral blood-derived origin of myeloid cells contributing to homeostatic turnover of eye macrophages.Figure 5a) Left, Experimental setup of surgically connected parabiotic mice. Acta1GFP/+ and Acta1+/+ mice underwent parabiosis for 2, 12 and 20 weeks before analysis. Right, quantification of GFP+Iba1+ microglia in the retina (rMG, squares, n.d. = not detectable), ciliary body (cbMΦ, triangles, Mann-Whitney ns p > 0.05) and cornea (cMΦ, circles, Kruskal-Wallis **p = 0.0024) of parabiotic mice. Blood chimerism of CD45+CD11b+Ly6ChiGFP+ cells in the analyzed wildtype mice was 37.7 ± 3.2 % (2 weeks), 27.5 ± 2.7 % (12 weeks) and 34.7 ± 3.7 % (20 weeks). Symbols represent mean ± s.e.m of three (2 weeks), four (12 weeks) or five (20 weeks) individual mice. Scale bars represents 50 µm. Representative immunofluorescence images from the retina (20 weeks) from flat mounts of Acta1+/+ parabiotic mice. GFP+Iba1+ double-positive cells are marked by arrows, GFP-Iba1+ single-positive cells are labelled by asterisks and GFP+Iba1- leukocytes are indicated by arrow heads. Pictures are representative of three animals. Representative immunofluorescence images from the , ciliary body (12 weeks) and cornea (20 weeks) from flat mounts of Acta1+/+ parabiotic mice. GFP+Iba1+ double-positive cells are marked by arrows, GFP-Iba1+ single-positive cells are labelled by asterisks and GFP+Iba1- leukocytes are indicated by arrow heads. Pictures are representative of three animals. e) Flow cytometric quantification of RFP+ cells in Ccr2-RFP mice among CD45+CD11b+CD115-Ly6Cint granulocytes (triangles, n = 4), CD45+CD11b+CD115+Ly6Clo monocytes (filled squares, n = 4 mice), CD45+CD11b+CD115+Ly6Chi monocytes (open squares, n = 4 mice), CD45loCD11b+ rMG (squares, n = 6 mice), CD45+CD11b+CD64+F4/80+ cbMΦ (triangles, n = 6 samples from twelve mice) and CD45+CD11b+CD64+F4/80+ cMΦ (filled circles, n = 12 mice, unpaired t test ***p < 0.0001). Data were obtained from one (rMG) or two independent experiments (blood, cbMΦ, cMΦ). Data are presented as means ± s.e.m. Flow cytometry of eye macrophages from healthy Ccr2RFP/+ mice (left) and representative histograms (right, Ccr2RFP/+ solid red line, Ccr2+/+ controls dotted grey line). Flow cytometry of eye macrophages from healthy Ccr2RFP/+ mice (left) and representative histograms (right, Ccr2RFP/+ solid red line, Ccr2+/+ controls dotted grey line). i) Flow cytometry of myeloid blood cells from Ccr2RFP/+ mice. Left: CD45+CD11b+ cells were further subdivided according to the expression of Ly6C and CD115 into CD45+CD11b+CD115-Ly6Cint granulocytes (gate 1), CD45+CD11b+CD115+Ly6Chi inflammatory (gate 2) and CD45+CD11b+CD115+Ly6Clo resident monocytes (gate 3), respectively. Right: representative histograms are shown (Ccr2RFP/+ solid red line, Ccr2+/+ controls dotted grey line). Four mice were investigated. j) Top left, sketch of Ccr2-RFP construct. Top right, typical confocal picture for CCR2 (red), Iba1 (green) and collagen IV (Coll IV, white) revealing Ccr2-RFP expression in a blood vessel (arrow, left image, scale bar represents 20 µm) and no RFP signal in retinal microglia (right images, scale bar represents 100 µm).
Figure 1Effects of basic and bulky hydrophobic amino acids on LT-induced pyroptosis and caspase-1 activation in RAWRA cells. Cells were pre-treated with 10 mM indicated amino acids prior to LT stimulation. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (A). Mock, DMEM medium. Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin antibody (B). p10, mature caspase-1; p45, caspase-1 precursor. C, D Effects of basic and bulky hydrophobic amino acids on LT-induced RFP-ASC specks formation in RAWRA cells. Fluorescence images were taken on a confocal microscopy (C). The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D). E Effects of basic and bulky hydrophobic amino acids on LT-induced caspase-1 activation in Tlr4-/- iBMDMs. Cells were treated as that in (A). Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin or anti-MEK3 antibody.
PolySer accumulation increases with age and severity of disease A Representative images of the vestibular nucleus (upper panels), cuneate nucleus (middle panels) and motor cortex layers II/III (lower panels) of SCA8 BAC mice at 2 months (left panels), 6 months (middle panels) and end-stage (10 months, right panels) stained with α-SerCT.Figure 3A Representative images of the vestibular nucleus (upper panels), cuneate nucleus (middle panels) and motor cortex layers II/III (lower panels) of SCA8 BAC mice at 2 months (left panels), 6 months (middle panels) and end-stage (10 months, right panels) stained with α-SerCT. Representative aggregates are indicated by black arrows. Corresponding quantifications of polySer aggregates for each region on the right (n=3; mean ± SEM; One-way ANOVA with Tukey's post-hoc test; * p<0.025, ** p<0.005, *** p<0.0005, **** p<0.0001) B Open field analysis of 2 month old animals show a significant decrease in ambulatory distance (cm) and ambulatory time (s) and a significant increase in resting time (s) (NT n=19, SCA8 BAC n=23, ** p<0.01, *** p<0.001; mean ± SEM; unpaired t test).
Figure 5Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. Dot plots of the percentage of Ki67+ proliferating CD8 T cells after ex vivo activation by A549-SC3 cells. CD8 T cells were obtained from samples of G1 or G2 patients before immunotherapy and after three cycles of anti-PD-1 therapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. Green, objective responders (OR) and red, no ORs. Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. but in the presence of an isotype control antibody or an anti-PD-1 antibody molecularly equivalent to pembrolizumab. Relevant statistical comparisons are shown with comparisons carried out with paired Student´s t test. Change in CD8 CD28+ T cells from baseline to post-therapy in G1 patients (left) or in G2 patients (right). Statistical comparisons were carried out with paired Student´s t test.
Figure 7A Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. *: p<0.05 by ANOVA with Tukey's post hoc test B Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hours C Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, and depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test D ALP staining and quantitative measurement of ALP activity. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 5 days. Results are shown as mean ± SEM; n=8; **: p<0.01 by ANOVA with Tukey's post hoc test  E Representative images of ARS staining and quantification. Cells were cultured with normal medium supplemented with 100 ng/ml BMP2 for 14 days. Results are shown as mean ± SEM; n=8; *: p<0.05, and **: p<0.01 by ANOVA with Tukey's post hoc test  F Representative images of ALP and ARS staining. Mouse MSCs were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 and 10 days, respectively G, H Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test  I, J Quantitative RT-PC analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test western blot analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test
Figure 1(A) Venn diagram of exon-exon junctions in 6,642 common genes identified among the Nanopore reads from the smg-2 (yb979) mutant and N2 as well as those deduced from gene models in WormBase (WS271). (B) Volcano plot of 8,701 mRNA isoforms from 2,931 genes with multiple isoforms. X-axis indicates a difference in percentage of each mRNA isoform within the gene (Δ%smg-2-N2). Y-axis indicates -log10 of p-value in Fisher's exact tests. Blue symbols indicate 219 mRNA isoforms significantly depleted from the smg-2 mutant (p<0.05, Δ%smg-2-N2<-10). Red symbols indicate 420 isoforms significantly enriched in the smg-2 mutant (p<0.05, Δ%smg-2-N2>10). (C, D) Length distribution of 3'UTRs (C) and proportions of putative PTC-containing isoforms (D) in 10,136 mRNA isoforms detected in N2 and/or smg-2 (All), the 219 isoforms depleted from and the 420 isoforms enriched in the smg-2 mutant. (E) GO terms significantly enriched in 259 genes with smg-2-enriched PTC isoforms (top) and in 30 genes with smg-2-depleted PTC isoforms (bottom). Modified Fisher's exact p-values in the DAVID Bioinformatics system are indicated. Note that 14 of the 30 genes with the smg-2-depleted mRNA PTC isoforms are common with those having smg-2-enriched PTC isoforms. This is why the same GO terms are enriched in these two groups. The mRNAs from such genes likely contain upstream open reading frames (uORFs), which may not be directly involved in or differentially affect NMD.
Prdx4 is secreted upon activation of the NLRP3 inflammasome and co-localizes with caspase-1 in MVBs A Western blot analysis of Prdx4, pro-caspase‑1, Gapdh and E-Cadherin from the cytosolic and insoluble cell fraction of LPS and/or ATP-stimulated BMDMs or untreated controls.Figure 5Western blot analysis of Prdx4, pro-caspase‑1, Gapdh and E-Cadherin from the cytosolic and insoluble cell fraction of LPS and/or ATP-stimulated BMDMs or untreated controls. Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs, primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP. Each circle represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test). Concentration of Prdx4 in the serum of WT mice, injected with LPS (4.5 mg/kg BW) for the time points indicated. Each dot represents an individual mouse. Horizontal lines indicate mean. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test). Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs. Cells were primed with LPS (100 ng/ml) for 6 h, followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation. Each bar represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test). Schematic illustration of selected mechanisms that were targeted by either Glycine, Necrosulfonamide (NSA) or GW4869 to study LPS+ATP-induced Prdx4 secretion. Relative levels of Prdx4 secretion in response to LPS+ATP stimulation and pretreatment with either Glycine, NSA or GW4869 and relative levels of caspase-1 secretion in response to LPS+ATP stimulation and pretreatment with in response to LPS+ATP stimulation and pretreatment with GW4869. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; n.s. not significant (two-tailed t-test). Western blot analysis of subcellular organelle fractions. OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS (100 ng/ml) for 12 h and stimulated with 5 mM ATP for 4 h.
Localisation of SCOC with the Golgi, Atg9 and LC3.Figure 3(A) Schematic of SCOC constructs. Numbers represent amino acids; CC is coiled‐coil domain. SCOC-myc was constructed with clone AF330205 (Van Valkenburgh et al, 2001). SCOC-GFP, GFP-SCOC and FLAG-SCOC were constructed with DNA corresponding to Uniprot isoform Q9UIL1‐3, Entrez gene isoform 5. (B) Endogenous SCOC shows colocalisation with Atg9 (red) and TGN46 (blue). HEK293 cells were fixed and permeabilised in saponin. Colocalisation indicated with arrows. (C) Endogenous SCOC (green top, red bottom) partially colocalises with endogenous LC3 (red, top) and GFP-LC3 (green, bottom) in HEK293 and GFP-LC3‐HEK cells, respectively, after 2 h amino‐acid starvation. Boxed region is at higher magnification in merge panel on right. Scale bars=10 μm.