EMBO/soda-nel huggingface dataset:
Circadian time- and sleep-dependent modulation of cortical parvalbumin-positive inhibitory neurons (2022)
Figure 6

Figure 6. PV neurons' daily activity rhythm relies on visual experience.

A. Diagram illustrating the timeline for repetitive imaging at ZT4, 4 hours sleep deprivation from ZT0 to ZT4 (SD_ZT4), and 4 hours sleep deprivation in darkness (Dark_SD). B. Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at ZT4 and SD_ZT4. The color code represents the magnitude of ΔF/F0 (%). C. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at ZT4 and SD_ZT4 (K-S test; P = 0.0001). Inset: the mean sum ΔF/F0 (ZT4: 322.4 ± 18.64; SD_ZT4: 424.0 ± 21.54; P = 0.0004; Mann-Whitney U test). D. Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at SD_ZT4 and Dark_SD. The color code represents the magnitude of ΔF/F0(%). E. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at SD_ZT4 and Dark_SD (K-S test; P = 0.015). Inset: the mean sum ΔF/F0 (SD_ZT4: 375.1 ± 20.19; Dark_SD: 293.8 ± 16.55; P = 0.0022; Mann-Whitney U test).

Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.
Circadian time- and sleep-dependent modulation of cortical parvalbumin-positive inhibitory neurons (2022)
Figure 7

Figure 7. Pyramidal neurons have opposite daily oscillation and dLGN-evoked response is negatively correlated with PV's activity.

A. Top panel, Example spontaneous ΔF/F0 traces from three putative pyramidal neurons repetitively imaged at ZT0 and ZT12. Bottom panels, Heatmaps showing the spontaneous ΔF/F0 time-series traces of all putative pyramidal neurons imaged at ZT0 and ZT12. B. The cumulative distribution of the sum ΔF/F0 of putative pyramidal neurons judged by skewness (K-S test; P < 0.0001). Inset: the means of sum ΔF/F0 (ZT0: 679.5 ± 38.22; ZT12: 485.8 ± 32.22; P < 0.0001; Mann-Whitney U test). C. Mean activity of putative pyramidal neurons indicated by Z score imaged at four timepoints. D1. Schematic showing in vivo dLGN-evoked Ca2+ reponses in V1. D2. Immunofluorescent staining showing the GCaMP6s (green) and ChR2-mCherry (red) expression in V1 and dLGN respectively. The optical fiber track was also labeled. (Scale: 500 µm). D3. Response profile of an example neurons for dLGN stimulation during the light/dark cycle. Horizontal blue brief lines represent stimulus frequency. E-F. Top: color-coded calcium signals of all responsive neurons (% ΔF/F0). Bottom: The mean Ca2+ response waveform for individual mouse (dotted color lines) and all mice (thick black and gray lines). Trials were aligned to LED onset time (gray solid lines). G. The fraction of neurons responsive to dLGN stimulus in ZT0 and ZT12. Each gray line represents one imaging mouse. Black and white circles and error bars represent the mean and SEM (ZT0: 8.51 ± 1.86; ZT12: 5.96 ± 1.39; P = 0.015; Wilcoxon signed-rank test; n = 9 mice). H. The Response fidelity of each cell in ZT0 and ZT12. Black and white circles and error bars represent the mean and SEM (ZT0: 0.42 ± 0.022; ZT12: 0.31 ± 0.023; P < 0.0001; Wilcoxon signed-rank test; n = 195 cell). I. Comparison of peak evoked response of each cell in ZT0 and ZT12. Black and white circles and error bars represent the mean and SEM (ZT0: 81.01 ± 4.79; ZT12: 48.42 ± 3.82; P < 0.0001; Wilcoxon signed-rank test; n = 195 cell).

Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.
Residual Pluripotency is required for Inductive Germ Cell Segregation (2021)
Figure 3

Figure 3. T directly regulates pluripotency factors

A Left: ChIP-seq tracks of T (in red; input is in blue) around the Oct4 (7.5 kb). DE, distal enhancer; PE, proximal enhancer. The T-binding peak around the Oct4-DE is indicated (arrowhead). Right: ChIP-qPCR for the enrichment of T on the DE and the regions indicated in Fig EV3E. NC, negative control (the locus without T binding). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates).

B Luciferase assay with T-binding elements (TBEs) around the indicated loci as shown in (A) and Fig EV3E. Relative luciferase activities of sorted cell fractions are shown with SDs. (three independent experiments with two technical replicates).

C Left: PCA of global gene expression in low T-inducible ZHBTc4 ESCs stimulated as indicated in Fig EV3G. Right: Heatmap of selected key pluripotency genes derived from mean-centered expression values (log2 scale).

D Top: Venn diagrams showing the overlap between down- and upregulated DEGs induced by both high T induction and Oct4 deletion in low T-activated condition (fold-change > 1; p < 0.1 by DESeq2). Bottom: Ontology terms enriched in overlapped DEGs. See also Dataset EV3.
Residual Pluripotency is required for Inductive Germ Cell Segregation (2021)
Figure 4

Figure 4. T activates the germ cell differentiation program in parallel with the disruption of naïve pluripotency

A Heatmap of k-means clustering derived from mean-centered expression values (log2 scale) of DEGs selected from 6000 DEGs (Fig EV4A) obtained from RNA-seq (Fig 2). See also Dataset EV1.

B FACS analysis of the induced fusion protein (T-V5-GR-2A-H2BGFP) and Blimp1-RFP reporter. Right: Measurements of Blimp1-RFP expression in three different cell fractions are also plotted on the histogram image.

C Unsupervised hierarchical clustering (correlation-based distance) obtained from RNA-seq in T-induced PGCLCs (T-PGCLCs), in vivo PGCs, and FACS-sorted cell fractions as indicated in Fig 2A-C.

D PCA of global gene expression in the same dataset as (D).

E Left: A representative confocal image of a mouse embryo at E7.0 bearing T-GFP and Blimp1-RFP transgenic reporters. Scale bar: 50um. Arrowheads indicate Blimp1-positive nascent germ cells in the posterior proximal epiblast. Right: FACS analysis of dissociated mouse embryos without visceral endoderm.

F Expression of the indicated genes measured by qPCR in FACS-sorted cells (E) derived from embryos at different developmental stages (E7.0-7.5). Ten FACS-sorted cells in different cell fractions (E) were used for each individual qPCR analysis with two technical replicates. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (n≥3, log2 scale). P values were calculated by two-tailed unpaired t-test.
Residual Pluripotency is required for Inductive Germ Cell Segregation (2021)
Figure 5

Figure 5. Coordinated regulatory mechanism of T and residual pluripotency factors is critical for a germ cell differentiation program

A ChIP-seq tracks of T (in red; input is in blue) and core pluripotency factors (in green) around the Blimp1 (100 kb) and Prdm14 (100 kb). The co-bound locus (L#1-4) and the locus exclusively bound by core pluripotency factors but not T (L#5) are indicated by arrow. NC, negative control (the locus without T binding).

B ChIP-qPCR for the enrichment of the indicated factors on the loci (L#1-5) indicated in (A). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates).

C Luciferase assay with the specific sequence of the locus bound by T around L#1 and L#3 as shown in (A). Relative luciferase activities of low T-inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs. (three independent experiments with two technical replicates).

D Expression of Blimp1 and Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (log2 scale, the mean value of two independent experiments with two technical replicates).

E Top: Oct4-GFP (green) reporter expression (left), and Blimp1-RFP (red) and Oct4-GFP (BROG) reporter expression (right) in the gonads of E13.5 embryos generated by 4N complementation with the indicated ESC lines. Scale bar: 100um. Bottom: percentage of Oct4-GFP reporter-positive PGCs (left) and BROG reporter-positive PGCs (right) measured by FACS analysis. Blue circles, red squares, and green triangles represent the percentage of gonadal PGCs in the first, second, and third independent mutant line, respectively, of the examined locus (L#1-5).

F Model indicating the inductive germ cell segregation achieved by a co-regulatory mechanism of residual pluripotency regulators and lineage regulators.

Expanded View Figure Legends
SATB2 Drives Glioblastoma Growth by Recruiting CBP to Promote FOXM1 Expression in Glioma Stem Cells (2020)
Figure 1

Figure 1. SATB2 is preferentially expressed by GSCs.

A. Immunofluorescence of SATB2 (green) and the GSC marker SOX2 or OLIG2 (red) on frozen tissue sections of human GBM surgical specimens. SATB2 is preferentially expressed by GSCs in human GBMs. Scale bar, 25 μm.

B. Quantification of the fraction of SATB2+ cells in SOX2+ or OLIG2+ cells in human GBMs. More than 90% SOX2+ or OLIG2+ cells showed SATB2 staining. n=3 GBMs.

C. qPCR analysis of SATB2 mRNA expression in GSCs and matched non-stem tumor cells (NSTCs) (n=5).

D. Immunoblot analysis of SATB2, SOX2 and OLIG2 expression in cell lysates of GSCs and matched NSTCs.

E. Immunofluorescenc of SATB2 (green) and SOX2 (red) in T3359 GSCs and matched NSTCs. Scale bar, 50 μm.

F. qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells (NPCs) (n=3).

G. Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs.

H. Immunoblot analysis of SATB2, GSC marker SOX2 and differentiation marker GFAP expression during serum-induced GSC differentiation.

Data information: Data are represented as mean ± SD. **p＜0.01, ***p＜0.001, Mann-Whitney test (C) or one way ANOVA analysis followed by Tukey's test (F). Exact p values are reported in Appendix Table S3.
SATB2 Drives Glioblastoma Growth by Recruiting CBP to Promote FOXM1 Expression in Glioma Stem Cells (2020)
Figure 2

Figure 2. Disrupting SATB2 impaired GSC proliferation and self-renewal.

A. Immunoblot analysis of SATB2 expression in GSCs transduced with lentiviral-mediated non-targeting shRNA (shNT) or SATB2 shRNA (shSATB2).

B. Cell viability of GSCs transduced with shNT or shSATB2 (n=5).

C. EdU incorporation assay of GSCs transduced with shNT or shSATB2. Scale bar: 50 μm.

D. Quantification of (C) showing the percentage of EdU+ cells (n=5).

E. Tumorsphere images of GSCs transduced with shNTor shSATB2. Scale bar: 100 μm.

F, G. Quantification of the diameter (F) or number (G) of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).

H. In vitro limiting dilution analysis of the tumorsphere formations of GSCs expressing shNT or shSATB2. Silencing SATB2 attenuated the self-renewal capacity of GSCs.

Data information: Data are represented as mean ± SD. ****p＜0.0001, two way ANOVA analysis followed by Tukey's test (B), one way ANOVA analysis followed by Tukey's test (D, F and G) or ELDA analysis for differences in stem cell frequencies (H). Exact p values are reported in Appendix Table S3.
SATB2 Drives Glioblastoma Growth by Recruiting CBP to Promote FOXM1 Expression in Glioma Stem Cells (2020)
Figure 3

Figure 3. Disrupting SATB2 inhibited tumor growth and prolonged mice survival.

A. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth.

B. Quantification of the bioluminescence of xenografts derived from the luciferase-labeled T3359 GSCs expressing shNT or shSATB2 on day 21 posttransplantation (n=5 mice per group).

C. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 (shNT: n=7 mice; shSATB2-1 or shSATB2-2: n=5 mice). Median survival: shNT, 28 days; shSATB2-1, 48 days; shSATB2-2, 45 Days. Animals bearing GSC-derived xenografts expressing SATB2 shRNA survived longer than the control animals.

D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.

E. Quantification of SATB2 intensity in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).

F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.

G. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).

H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm.

I. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group).

Data information: Data are shown as mean ± SD. *p＜0.05, ***p＜0.001, ****p＜0.0001 compared with shNT group, Kruskal-Wallis test followed by Dunn's test (B), log-rank test (C) or one way ANOVA analysis followed by Tukey's test (E, G and I). Exact p values are reported in Appendix Table S3.
SATB2 Drives Glioblastoma Growth by Recruiting CBP to Promote FOXM1 Expression in Glioma Stem Cells (2020)
Figure 4

Figure 4. SATB2 regulates cell cycle gene expression.

A. Heatmap analysis of differentially expressed genes between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT). Differentially expressed genes had a 1.8-fold or greater expression difference. Among differentially expressed genes, 160 are upregulated and 185 are downregulated.

B. Gene ontology analysis of genes downregulated in SATB2 silencing GSCs compared with control GSCs. The most significantly down-regulated genes are associated with cell cycle progression and chromosome organization. p values were generated using the PANTHER tool (http://pantherdb.org/).

C. qPCR analysis of FOXM1 and SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 (n=3).

D. Immunoblot analysis of FOXM1 and SATB2 expression in GSCs transduced with shNT or shSATB2.

E. Immunoblot analysis of FOXM1 and SATB2 expression in tumor xenografts-derived from T3359 GSCs expressing shNT or shSATB2.

F. Heatmap analysis of FOXM1 downstream targets between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT) from microarray analysis. Differentially expressed genes had a 1.5-fold or greater expression difference.

G. qPCR analysis of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 (n=3).

Data information: Data are represented as mean ± SD. *p＜0.05, **p＜0.01, ***p＜0.001, ****p＜0.0001, one way ANOVA analysis followed by Tukey's test. Exact p values are reported in Appendix Table S3.
SATB2 Drives Glioblastoma Growth by Recruiting CBP to Promote FOXM1 Expression in Glioma Stem Cells (2020)
Figure 5

Figure 5. Ectopic expression of FOXM1 rescued the decreased GSC proliferation and GBM growth caused by SATB2 disruption.

A. Immunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2.

B. Cell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing.

C. Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing.

D, E. In vivo bioluminescent images (D) or quantification (E) of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.

F. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=8 mice; shNT+FOXM1: n=7 mice; shSATB2: n=6 mice; shSATB2+ FOXM1: n=8 mice). Median survival: shNT, 30 days; shNT+FOXM1, 28 days; shSATB2, 50.5 Days; shSATB2+FOXM1, 34 days. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC-derived GBMs.

G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.

H. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).

I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.

J. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).

Data information: Data are shown as mean ± SD. **p＜0.01, ***p＜0.001, ****p＜0.0001, two way ANOVA analysis followed by Tukey's test (B), one way ANOVA analysis followed by Tukey's test (C, E, H and J) or log-rank test (F). Exact p values are reported in Appendix Table S3.
SATB2 Drives Glioblastoma Growth by Recruiting CBP to Promote FOXM1 Expression in Glioma Stem Cells (2020)
Figure 6

Figure 6. SATB2 binds to the MAR sequence in the FOXM1 gene locus and recruits CBP acetyltransferase in GSCs.

A. Schematic representation of MAR (Matrix Attachment Region) within FOXM1 locus. Arrows show the location of PCR primers for ChIP experiments.

B. ChIP assays with the SATB2 antibody or IgG using GSCs, NSTCs and NPCs. PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Note that abundant SATB2 binds to the MAR of FOXM1 gene locus in GSCs.

C. qPCR analysis of ChIP assays with the SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 decreased its binding amount to the MAR of FOXM1 gene locus.

D. CoIP assays of endogenous protein interaction in GSCs. Immunoblots of precipitated proteins or total lysates were performed using indicated antibodies. Note that SATB2 associates with endogenous CBP while not P300.

E. qPCR analysis of ChIP assays with the CBP antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced the binding of CBP to the MAR of FOXM1 gene locus.

F. qPCR analysis of ChIP assays with the indicated antibody (AcH3K18, AcH3K27, AcH4) using T3359 GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced acetylation of H3K18, H3K27 and H4 levels on the MAR of FOXM1 locus.

G. qPCR analysis of FOXM1 mRNA expression in T3359 GSCs transduced with shSATB2 or shCBP or both (n=3).

H. Immunoblot analysis of FOXM1 expression in T3359 GSCs transduced with shSATB2 or shCBP or both.

Data information: Data are shown as mean ± SD. *p＜0.05, **p＜0.01, ***p＜0.001, ****p＜0.0001, one way ANOVA analysis followed by Tukey's test. Exact p values are reported in Appendix Table S3.
SATB2 Drives Glioblastoma Growth by Recruiting CBP to Promote FOXM1 Expression in Glioma Stem Cells (2020)
Figure 7

Figure 7. C646 treatment inhibited GSC proliferation and GSC-driven tumor growth.

A. Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control (n=5).

B. Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days. Scale bar: 100 μm.

C. qPCR analysis of SATB2, FOXM1 and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 hours (n=3).

D. Schematic diagram showing the treatment of mice bearing the GSC-derived xenografts with C646. After GSC transplantation for 7 days, mice were treated with C646 or the vehicle control daily. Mice were monitored by IVIS bioluminescent imaging and maintained until neurological signs occur.

E. Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase-labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation (n=5 mice per group).

F. Quantification of tumor growth from (E) (n=5 mice per group).

G. Kaplan-Meier survival curves of mice bearing T3359 GSC-derived xenografts treated with C646 or the vehicle control (Vehicle control: n=9 mice; C646: n= 8 mice). Median survival: Vehicle control, 27 days; C646, 33.5 days.

H. Immunofluorescence of Ki67 (Green) in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). Scale bar: 40 μm.

I. Quantification of Ki67 positive cells in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group).

Data information: Data are represented as mean ± SD. *p＜0.05, **p＜0.01, ***p＜0.001, ****p＜0.0001, two way ANOVA analysis followed by Tukey's test (A), one way ANOVA analysis followed by Tukey's test (C), two way ANOVA analysis followed by Sidak's test (F), log-rank test (G), or Mann-Whitney test (I). Exact p values are reported in Appendix Table S3.
LRRK2 localizes to endosomes and interacts with clathrin-light chains to limit Rac1 activation (2015)
Figure 1

Figure 1. Identification of CLCs as LRRK2-binding partners.

A. Equal aliquots (2 mg) of a Triton X-100 solubilized lysate from rat brain were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.

B. Brain lysate as in (A) was incubated with GST or GST-nCLCb. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.

C. Full-length GST-nCLCb (1-228) and various truncation mutants were purified from bacteria. Equal aliquots of purified protein were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibody against CLCs.

D. Brain lysate as in (A) was incubated with GST or GST-nCLCb proteins with boundaries as indicated. Specifically bound proteins were detected by Western blot with antibody against CLCs.

E. Representation of a triskelia composed of three CHCs (orange) with three associated CLCs (blue).

F. Highly enriched triskelia were stripped from purified CCVs. The triskelia were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins. For A-D/F, aliquots of the starting material (SM) equal to 10% of that added to the beads, was analyzed in parallel.