EMBO/soda-nel huggingface dataset:
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 5

Figure 5. Ste11 and Ssk2/Ssk22 differentially phosphorylate Pbs2

A-B. Detection of Pbs2 phosphorylation at S514 and T518. The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (A) Phos-tag band-shift assay or (B) anti-phospho-T518 immunoblotting. In (A), the positions of Pbs2-HA phosphorylated (S514-P) and unphosphorylated (S514-OH) at S514 are indicated.

C-F. NaCl dose-response analyses of Pbs2 phosphorylation. (C-D) KT005 (ste11Δ pbs2Δ) and (E-F) TM280 (ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. (C and E) S514 phosphorylation was analyzed using the Phos-tag band-shift assay. PC; Positive Control. (D and F) T518 phosphorylation was analyzed using anti-phospho-T518 immunoblotting.

G-H. Average values of three independent experiments from (C-D) and (E-F), respectively, were plotted. AU, arbitrary unit.

I. Detection of di-phosphorylated Pbs2. KT005 (ste11Δ pbs2Δ) transformed with YCplac22I'-Pbs2-HA was treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extracts, and subjected to Phos-tag SDS-PAGE. Blots of these gels were probed with (upper panel) anti-phospho-T518 or (lower panel) anti-HA.

J. Same as in (I), except that the yeast strain KT003 (pbs2Δ) was used.

Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 6

Figure 6. Mono-phosphorylated Pbs2 can phosphorylate Hog1 only when the Pbs2-Hog1 reaction is enhanced

A. The Pbs2 single and double Ala substitution mutations used in this study.

B. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. KT005 (ste11Δ pbs2Δ) was transformed with YCplac22I'-Pbs2 -HA (WT) or its indicated mutant derivatives. Cells were stimulated with (+) or without (-) 0.6 M NaCl for 5 min.

C-F. Phos-tag band-shift analyses of Hog1 phosphorylation. KT291 (ste11Δ pbs2Δ hog1Δ) was transformed with (C and D) pRS416-Hog1 or (E and F) pRS416-Hog1-ΔL16, together with (C and E) YCplac22I'-Pbs2-S514A-HA or (D and F) YCplac22I'-Pbs2-T518A-HA. Cells were stimulated with the indicated concentrations of NaCl for 5 min.

G-H. Average values of three independent experiments from (C and E) and (D and F), respectively, are plotted.

I. A scheme illustrating the osmotic enhancement of the Pbs2-Hog1 reaction in activating Hog1 by mono-phosphorylated Pbs2.

Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 7

Figure 7. The Hog1-N149H/D162G mutant constitutively enhanced the Pbs2-Hog1 reaction

A Phos-tag band-shift assay of Hog1 phosphorylation. KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) was transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives. Cell extracts were prepared from fresh cultures without applying osmostress. For each HOG1 mutant plasmid, three independent cultures were assayed.

B. Same as in (A), except that the yeast strains KT260 (ssk2/22Δ ste11Δ hog1Δ), KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P), and KT248 (ΔS/O/H/M ssk2/22Δ hog1Δ pbs2Δ) carrying YCplac22I'-Pbs2-DD, were used.

C. KT250 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-WT) and KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) were transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N149H D162G) together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed.

D. KT248 (ΔS/O/H/M ssk2/22Δ hog1Δ pbs2Δ) was transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed.

E-F. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strains of the indicated genotypes (shown below the graph) were transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives (shown inside the graph). Strain used: (E) FP4, KT259, KT260, KT292, and KY523; and (F) KY523, KT293, KT294, and KT295. N/H, N149H; D/G, D162G.

G. A schematic model showing that the lack of osmotic enhancement of the Pbs2-Hog1 reaction prevents the basal Hog1 activation.

Data information: (A-B and D-F) Error bars are SEM (n=3). (C) Error bars are SEM (n=4).
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 8

Figure 8. The yeast pheromone-to-Hog1 crosstalk and the basal activity of the mammalian p38 MAPK might be suppressed by the same mechanism

A. KT299 (MATa hkr1Δ msb2Δ ssk2/22Δ) was exposed to the indicated concentrations of α-factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting.

B. KT299 (MATa ssk2/22Δ hkr1Δ msb2Δ) was exposed to the indicated concentrations of α-factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay.

C. Average values of four independent experiments from (B) were plotted.

D. HM06-1 (MATa ΔS/O/H/M ssk2/22Δ) was exposed to the indicated concentrations (log scale) of α-factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average values of three or more independent experiments were plotted.

E. KT306 (MATa hkr1Δ msb2Δ ssk2/22Δ hog1Δ) was transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N/H D/G), and was exposed to 10 μM α-factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average of three independent experiments are plotted.

F. A schematic model showing that the lack of osmotic enhancement of the Pbs2-Hog1 reaction prevents the pheromone-to-Hog1 crosstalk.

G. Typical FRET (YFP/CFP ratio) images showing p38 activation. HeLa cells carrying the p38 reporter PerKy-p38 (Tomida et al., 2015) were stably transfected with an expression vector for the indicated p38α mutant proteins. FRET analysis was performed as described in Materials and Methods.

H. Distribution of p38 activity in individual cells from sets (a) - (d) in (G).

Data information: (B) Representative results from three independent experiments. (C-E) Error bars are SEM: (C) n=4; (D), n=3 or more; (E) n=3. (G) Scale bars: 20 μm. (H) Statistics, Student's two-tailed t-test.
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 9

Figure 9. Time-courses and feedback regulation of the Hog1 phosphorylation

A. An example of the time-course experiments for the osmostress-induced Hog1 phosphorylation. The yeast strain KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) was stimulated with 1.0 M NaCl for the indicated times, and the percentage of phosphorylated Hog1 (Hog1-P) was determined using a Phos-tag band-shift assay.

B-D. Compilations of the time-courses of osmostress-induced Hog1 activation in (B) KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P), (C) TM257 (ssk2/22Δ), and (D) TM142 (wild-type). For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. The color chart below (D) indicates the concentrations of NaCl used.

E. Effects of Hog1 kinase activity and osmostress-induced glycerol accumulation on the time-courses of Hog1 activation. Cells were stimulated with 0.4 M NaCl (left panel) or 1.6 M NaCl (right panel) for the indicated times, and the percentage of phosphorylated Hog1 was determined using Phos-tag band-shift assay. Strains used were: TM142 (WT), KT254 (gpd1Δ gpd2Δ stl1Δ), and TM232 (hog1Δ) carrying pRS416-HOG1-K52S K53N. K/S K/N, K52S K53N; gpd1/2, gpd1Δ gpd2Δ.

F. A model of the negative and positive feedback regulations in the HOG pathway. The upstream osmosensors are Sln1 and the complexes composed of Sho1, Opy2, Hkr1, and Msb2. The molecular identity of the downstream osmosensor, which enhances the signaling between Pbs2 and Hog1, is currently unknown.

Data information: (B-D) For each data point, n=1 or more. (E) Error bars are SEM (n=3 or more).

Legends to Expanded View Figures
CBP/p300 and HDAC activities regulate H3K27 acetylation dynamics and zygotic genome activation in mouse preimplantation embryos (2022)
Figure 1

Figure 1. Dynamic landscape of H3K27ac in mouse oocytes and early embryos

Immunostaining of H3K27ac in mouse germinal vesicle (GV) oocytes (n = 8), metaphase II (MII) oocytes (n = 7), zygotes (n = 7), early 2-cell (2C) (n = 7) and late 2C (n = 9) embryos. Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages. Scale bar: 20 μm.

Genome browser view showing the landscape of H3K27ac in oocytes and early embryos.

H3K27ac domain size distribution at different stages.

Dynamic changes of H3K27ac from GV oocytes to zygotes. All the H3K27ac domains from GV to zygote were classified into three clusters (C1-C3) using k-means clustering. C: domain center.

Dynamic changes of H3K27ac from zygotes (before ZGA) to late 2-cell embryos (major ZGA). All the H3K27ac domains from zygotes to late 2-cell embryos were classified into three clusters (C1-C3) using k-means clustering.

Enrichment of H3K27ac at major ZGA genes, maternal decay genes and Polycomb group (PcG) targets at different stages. TSS: transcription start site; TES: transcription end site.

Data information: For boxplots in A and C, the central band represents the median. The lower and upper edges of the box represent the first and third quartiles, respectively. The whiskers of the boxplot extend to 1.5 times inter-quartile range (IQR).
CBP/p300 and HDAC activities regulate H3K27 acetylation dynamics and zygotic genome activation in mouse preimplantation embryos (2022)
Figure 4

Figure 4. CBP/p300 inhibition in zygotes leads to H3K27ac loss, ZGA failure and development arrest

Schematic illustration of transient CBP/p300 inhibition from zygote to early 2-cell with A-485. The cell cycle stages were based on (Abe et al., 2018). The arrows indicate the time points of sample collection for RNA-seq.

Immunostaining of H3K27ac in A-485 treated embryos versus control (DMSO treated) at different time points. Scale bar: 20 μm.

Images of embryos treated with DMSO (control) and A-485 at 96 hpi. Scale bar: 80 μm.

Developmental rate of embryos treated with A-485 versus control.

Scatter plots showing the whole transcriptome changes in zygotes, early 2-cell and late 2-cell embryos after CBP/p300 inhibition.

Scatter plots showing the expression level changes of zygote minor ZGA genes (n = 30), early 2-cell minor ZGA genes (n = 558) and major ZGA genes (n = 2,773) after CBP/p300 inhibition.

Scatter plot showing the expression level changes of maternal decay genes (n = 3,701) in early 2-cell and late 2-cell embryos after CBP/p300 inhibition.
CBP/p300 and HDAC activities regulate H3K27 acetylation dynamics and zygotic genome activation in mouse preimplantation embryos (2022)
Figure 6

Figure 6. HDAC inhibition blocks deacetylation from zygote to 2-cell embryos and leads to premature expression of developmental genes

Schematic illustration of HDAC inhibition from zygotes to late 2-cell embryos with TSA (Trichostatin A). The cell cycle stages were based on (Abe et al., 2018). The arrow indicates the time point of sample collection for RNA-seq.

Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control (DMSO treated) at 28 hpi. Scale bar: 20 μm.

Images of embryos treated with DMSO (control) or TSA at 96 hpi. Scale bar: 80 μm.

Developmental rate of embryos treated with TSA at different time points (0-20 hpi and 8-28 hpi) versus control.

Late 2-cell embryo whole transcriptome comparison between HDAC inhibition (TSA treated) and control (DMSO treated).

Heatmap showing the up-regulated genes after TSA treatment at late 2-cell and the corresponding H3K27ac dynamics at the promoter of these up-regulated genes.

Gene Ontology (GO) enrichment analysis of the up-regulated genes after HDAC inhibition.

Expanded View Figure legends
The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity (2022)
Figure 4

Figure 4. Human homologue of Ydj1, DnaJA1, modulates Abeta42 species formation and complements Abeta42 toxicity phenotypes in the absence of Ydj1. (A-B) Quantification of propidium iodide (PI)-staining positive cells indicating cell death at indicated time points of expression of EGFP-A42 (A42) Δydj1 cells with co-expression of DnaJA1-FLAG (DnaJA1) (B) or corresponding empty vector controls (ev) (A). Mean ± SD n = 4–6 biologically independent cultures. Comparisons by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus control). (C)Immunoblot (WB) of whole-cell extract (Input) and eluate of FLAG-tagged DnaJA1 immunoprecipitation (IP: FLAG) of wild-type strain (WT) expressing EGFP-A42 (A42) and co-expressing DnaJA1-FLAG (A1) using Abeta-specific antibody (6E10) and FLAG antibody (FLAG). (D) Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity of YDJ1 deletion strain (Δydj1) and wild type (WT) cells expressing EGFP-A42 (A42) only or co-expressing DnaJA1 was measured at two time points (t0 and t2) and normalized to t0. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2C. (E) Immunoblot (WB) of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.), and cytosolic (Cyt.) fractions of YDJ1-deleted cells (Δydj1) after 18 h expression of EGFP-A42 (A42) only or co-expressing DnaJA1-FLAG (DnaJA1) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time (Short exp.) exposure (two sections from one immunoblot). Cox4-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction. (F) Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 (A1) and quantified by densitometry in (G). (G) Quantification of the oligomer-to-monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in (F). Dot plots show all data points along with the mean (line) ± SD n = 3 biologically independent cultures. P-values by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus A42 control). See also Appendix Fig S2D. (H) A42 beta-sheet-rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ. Data represent means of at least eight measurements. See also Appendix Fig S1E.
The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity (2022)
Figure 5

Figure 5. Human homologue of Ydj1, DnaJA1, interacts with Abeta42 and its oligomers in 3xTg mouse model and is altered in post-mortem brain samples of AD patients.

(A) Immunoblot of cytosol-enriched cerebral tissue homogenate (Input) and eluate of Abeta42 or DnaJA1 using Abeta-specific antibody (6E10) and DnaJA1-specific antibody (DnaJA1), respectively, showing optimal and long exposure (Long exp.). Immunoprecipitation using magnetic beads without antibody, DnaJA1 or 6E10 antibody of female (15 months old) wild type (WT) or 3xTg (PS1M146V/APPSwe/tauP301L) mice. Sections showing monomer and low-n oligomers, dodecamer and full-length amyloid precursor protein (APP) are from 1 immunoblot.

(B) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). PonceauS serves as a loading control.

(C) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). Representative immunoblot is shown in (B). Dot plots show all data points along with the mean (bar) ± s.d.. n = 4-5 independent patients. *, p < 0.05. Unpaired, two-tailed t-test.
The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity (2022)
Figure 6

Figure 6. Droj2, fly homologue of DnaJA1, is upregulated in response to Abeta42 expression and its downregulation decreases neuronal Abeta accumulation.

(A) Representative confocal microscopy of 10-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42). See also Fig EV4A-B.

(B) Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 18-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).

(C-D) Average intensity (C) and total area (D) of Abeta (6E10) signal from confocal images representatively shown in (A) from 12 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 10-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 12. *, p < 0.05. Unpaired, two-tailed t-test. See also Fig EV4C-D.

(E-F) qPCR analysis of Droj2-mRNA levels of 3-6 days old female (E) and male (F) flies expressing human Abeta42 (UAS-A42) of w1118 wild type flies (Droj2+/+) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression (Droj2+/+ ctrl). Reference gene is Rpl32. Dot plots show all data points along with the mean (line) ± s.d.. n = 3. **, p < 0.01. One sample t-test against 1.

(G) Immunoprecipitation (IP: 6E10) of w1118 wild type flies (Droj2+/+) using synthetic Abeta42 added to the fly head extract (Input). Abeta-specific antibody 6E10 was used for Abeta42 and DnaJA1-specific antibody for Droj2 immunoblot detection. Showing input, supernatant, supernatant after washing (WS) and eluate. See also Fig EV4E.
The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity (2022)
Figure 7

Figure 7. Abeta42-induced mortality, cognitive impairment and mitochondrial morphology changes depend on the fly homologue of DnaJA1, Droj2.

(A-B) Survival of female (A) and male (B) w1118 wild type flies (Droj2+/+) and Droj2 knockdown flies (Droj2+/-) with expression of human Abeta42 (UAS-A42) or control flies without expression (ctrl), upon supplementation of sugar (10% sucrose) with 20 mM MnCl2. Survival was determined at indicated time points. n = 6 with 100-120 flies per experiment. The indicated p-value refers to the interaction (int.) term of a Cox Proportional Hazards model comparing Abeta42 toxicity (UAS-A42 vs. ctrl) and Droj2 expression (Droj2+/+ vs. Droj2+/-) as main factors. The following pairwise comparisons of the indicated groups survival were done by Log Rank test (****, p < 0.0001; ns, p > 0.05). See also Appendix Fig S1I-K.

(C) Aversive associative memory performance 2 min after training of aged (18 days old) male Droj2 knockdown flies (Droj2+/-) and corresponding isogenic w1118 wild type flies (Droj2+/+) both expressing human Abeta42 (UAS-A42) of six independent biological replicates. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6. *, p < 0.05. Unpaired, two-tailed t-test. See also Fig EV4G.

(D) Representative confocal and gSTED deconvolved images of Kenyon cells in 15-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A-specific antibody (ATP5A, green) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).

(E-F) Solidity (E) and circularity (F) of ATP5A-stained mitochondria from fly brain gSTED deconvolved images representatively shown in (D) from 8-10 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 15-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8-10. *, p < 0.05. Unpaired, two-tailed t-test. See also Fig EV5D-H.

Expanded View Figure legends
Mechanism and functions of membrane binding by the Atg5–Atg12/Atg16 complex during autophagosome formation (2012)
Figure 1

Membrane binding by Atg5–Atg12/Atg16 complex and its components. (A) Coomassie‐stained gels showing the results of liposome co‐sedimentation assays using Folch liposomes and the indicated proteins. Liposome binding allows the protein to be pelleted. The star indicates a degradation product of the Atg5–Atg12 conjugate. (B, D) Yeast cells of the indicated genotype were transformed with plasmids coding for Atg5–9 × Myc and Atg16–9 × Myc, respectively, and subjected to fractionation experiments. The proteins were detected by anti‐Myc western blotting. The presence of the protein in the pellet fraction indicated membrane binding. (C) Quantification showing the relative amounts of Atg5–9 × Myc and Atg5–9 × Myc–Atg12 in the pellet fractions relative to Pex30. The amount of Atg5–9 × Myc and Atg5–9 × Myc–Atg12 in atg5Δ cells was set to 1. See Supplementary Figure 1 for graph based on non‐normed data. The quantification is based on three independent experiments and the averages and the standard deviations are shown. The numbers next to the blots indicate the molecular weight in kDa. P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.
Mechanism and functions of membrane binding by the Atg5–Atg12/Atg16 complex during autophagosome formation (2012)
Figure 2

The lipid requirements for membrane binding by the Atg5–Atg12/Atg16 complex. (A) Coomassie‐stained gels of liposome co‐sedimentation assays using either Atg5–Atg12/Atg16 (upper panel) or Atg5/Atg16 (lower panel) and liposomes with the indicated lipid composition (Folch: Folch lipids, PE/PI3P: 40% POPC, 35% POPS, 20% POPE, 5% PI3P, PE/PI: 40% POPC, 35% POPS, 20% POPE, 5% PI, DAG/PI3P: 40% POPC, 35% POPS, 20% DAG, 5% PI3P, DAG/PI: 40% POPC, 35% POPS, 20% DAG, 5% PI). Note that the Atg5–Atg12/Atg16 and the Atg5/Atg16 complexes show almost identical binding behaviours. (B) Quantification showing the amounts of Atg5–Atg12/Atg16 and Atg5/Atg16 in the pellet fractions. The amount of protein in the pellet fraction in the absence of liposomes (background) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes. Data were obtained from three independent experiments, one of which is shown in (A). The Atg5–Atg12 and Atg5 bands were used for quantification. (C) Coomassie‐stained gel of a liposome co‐sedimentation assay showing the lipid requirements of the Atg5–Atg12/Atg16 complex. Liposomes were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. In the liposomes containing no POPE or POPS, these lipids were replaced with POPC. (D) Quantification of Atg5–Atg12/Atg16 in the pellet fraction in liposome co‐sedimentation, one of which is shown in (C). Data were obtained from six independent experiments. The Atg5–Atg12 was used for quantification. The average value is shown and the error bars represent the standard deviation. P‐values were calculated using Student's t‐test. The numbers next to the gels indicate the molecular weight in kDa. DAG, diacylglycerol; P, pellet; PI, phosphatidylinositol; PI3P, phosphatidylinositol‐3‐phosphate; (PO)PC, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylcholine; (PO)PE, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylethanolamine; (PO)PS, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylserine; S, supernatant. Figure source data can be found with the Supplementary data.