EMBO/soda-nel huggingface dataset:
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 2

Figure 2. A sequence motif within ub-hotspots is bound by Ymr111c/Euc1.

(A) Schematic of the ub-HS-motif mapping strategy. A 1038 bp stretch of ub-HS4 (blue line) was cloned and integrated at the LEU2 locus (grey) using the integrative YI128-vector (green). Initial mapping led to the identification of fragment F1 (Appendix Fig. S2A), which was further truncated for fine-mapping ((B), F5-F8). qPCR primers were designed to bind within the YI128 backbone.

(B) A 39 bp fragment of ub-HS4 is sufficient to drive ectopic ub-HS formation. Fragments of ub-HS4 were integrated ectopically and ub-K48 ChIP-qPCR was performed for the endogenous ub-HS5 and the ectopic ub-HS4 fragments as depicted in (A). contr.: control, empty YI128-vector was integrated at LEU2. Experiments were performed in cdc48‑6 background. Data represent means ± SD (n = 2).

(C) Experimental mapping and bioinformatic prediction identify a similar ub-HS-motif. Comparison between the experimentally mapped ub-HS4 F7 (B) and the consensus motif of all ub-HSs identified by the MEME software.

(D) A yeast one-hybrid screening strategy to identify proteins binding to the ub-HS-motif. Three copies of the ub-HS4-motif (F7) were cloned upstream of a minimal promoter followed by a HIS3 reporter-gene and integrated at the URA3 locus. A yeast cDNA library with N-terminally fused Gal4-AD was used to screen for survival on media lacking histidine and multiple plasmids coding for 4 different genes were recovered (bottom). See also Fig. EV1G.

(E) Euc1 binds to the ub-HS-motif in a Y1H assay. Gal4-AD- or Gal4-AD-Euc1-encoding plasmids were transformed into a Y1H reporter strain as described in (D). Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3-amino-triazole (3AT). Cells were grown at 30°C for 3 days.

(F) Euc1 is required for formation of ub-HSs. ChIP with ub-K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR. Data represent means ± SD (n = 4).

(G-H) Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of K48-linked ubiquitin chains (G) or Euc1 (H) were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 3

Figure 3. The transcription-factor like Euc1 shows transactivation in reporter gene assays.

(A) Euc1 domain structure is reminiscent of transcription factors. Top: Schematic representation of Euc1, with a predicted coiled-coil domain (CC) and GCR1-domain indicated. Predicted DNA binding of the C-terminal part was confirmed in Y1H experiments (Appendix Fig. S3A-C). Bottom: Euc1-like protein sequences from closely related Saccharomyces species were aligned and a phylogenetic tree was generated using Clustal Omega. Jalview was used to graphically display the degree of sequence conservation (see also Appendix Fig. S4A).

(B) The ub-HS-motif is conserved in closely related yeast species. The 7 yeast Multiz Alignment & Conservation tool of the UCSC Genome Browser was used to retrieve alignments of sequences corresponding to ub-HS-motifs from other Saccharomyces species. Dot (.) indicates a conserved base.

(C) Euc1 can induce transactivation via its N-terminal 30 amino acids. Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig. 2D and serial dilutions were spotted on control or selective media to test HIS3-activation. Cells were grown at 30°C for 3 days.

(D) Quantification of HIS3 mRNA levels from strains used in (C). Cells were grown in liquid media with selection for the transformed plasmids (SC-Leu), harvested in logarithmic growth phase and total mRNA was prepared. After reverse transcription, HIS3 mRNA levels were quantified using qPCR (RT-qPCR), normalized first to ACT1 mRNA and then to the empty vector control strain. Data represent means ± SD (n = 4). p = 2.43*10-5 (Student's t-test).
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 4

Figure 4. Euc1 is SUMOylated, recruits Slx5 to ub-hotspots, and is ubiquitylated in a Slx5/Slx8-dependent manner.

(A) Euc1 interacts with SUMO pathway proteins in a yeast two-hybrid (Y2H) assay. A Y2H reporter strain (PJ69-7a) was transformed with Gal4 DNA-binding domain (BD) and Gal4 activation domain (AD) fusion constructs in the indicated combinations. AD-SUMO-GG can be conjugated to SUMOylation substrates, while AD-SUMO-AA is conjugation-deficient. Cells were spotted on control media or selective media (- His) and grown for 3 and 6 days, respectively.

(B) Euc1 is SUMOylated by Ubc9, Siz1 and Siz2 on K231. Euc1 was immunoprecipitated from the indicated strains and eluates were probed by WB against SUMO (top) and Euc1 (bottom). An up-shifted band corresponding to Euc1SUMO was detected with both antibodies. Asterisks denote non-specific bands.

(C) Euc1 is predominantly monoSUMOylated on lysine 231. Denaturing NiNTA-pulldowns (NiNTA-PD) with strains expressing His-tagged SUMO (HisSUMO) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter. Covalently SUMO-modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1. Eluates were probed for SUMO to control for equal pull-down, Pgk1 was probed as input control. Euc1-KR denotes the K231R mutation here and hereafter.

(D) Euc1 SUMOylation is required for ub-HS formation. Ub-K48 ChIP quantified by qPCR for the indicated strains. The ubx5∆ background was used to enhance the ubiquitin signal, similar results were obtained in a WT background. Data represent means ± SD (n = 3).

(E) Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation. Denaturing NiNTA-PDs as described for (C) but with strains expressing His-ubiquitin (HisUbi) and 3FLAGEuc1 under the control of the ADH promoter. WBs were developed with a FLAG antibody to probe for 3FLAGEuc1, a ubiquitin blot served as PD-control and Dpm1 as input control. The slx8-CD allele carries the C206S, C209S-mutations (Xie et al, 2007).

(F-G) SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub-HSs. ChIP against Slx5 and Slx8 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and 3HA-Slx5 in (F) and WT and Slx8-9myc in (G) are from Fig. 1D and are shown here for comparison. Data represent means ± SD (n = 2).
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 5

Figure 5. Specific substrate-ligase interaction sites mediate SUMO-SIM independent Euc1-Slx5 binding.

(A) Euc181-183 binds to Slx5 in Y2H assays. Y2H assay performed as in Fig. 4A. Serial dilutions were spotted for each plasmid combination and cells were grown at 30°C for 2 days. The Slx5-RING∆ construct is deleted for the Slx5 C-terminus from beginning of the RING-domain (∆aa 488-619). See also Fig. EV3B.

(B) Euc1 binds Slx5 in vitro. Recombinant purified GST or GSTEuc1 were used in GST pull-down assays to co-precipitate recombinant 6HisSlx5.

(C) Euc1 binds to Slx5 in vivo. Cell lysates from an euc1∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids (under EUC1 promoter) were subjected to immunoprecipitation using anti-FLAG beads. IP-eluates were probed by WB with Slx5, SUMO and Euc1 antibodies, inputs were probed with Slx5, FLAG and Dpm1 antibodies (top to bottom). Note that the Euc1-Slx5 interaction is independent of the Slx8 ligase activity (slx8-CD, lanes 5-8).

(D) Euc1 Slx5-binding mutants (SBM1, SBM2) affect Euc1-Slx5 interaction in Y2H assays. AD-Euc181-183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD-Slx5 constructs as described in Fig. 4A. Mutations: SBM1: aa 139-143 ENQKK>ANAAA, SBM2: aa 162-165 KEVF>AAAA. Serial dilutions were spotted and cells were grown at 30°C for 2 days. See Appendix Fig. S6A for expression levels.

(E) Euc1-SBM constructs show defective Slx5 binding in vivo. Mutations described in (D) were introduced into full-length Euc1 constructs (with or without the K231R mutation) and FLAG IPs were performed as described in (C). WB analysis showed strong Slx5-binding defects for the SBM1/SBM2 and SBM1+2 constructs (top panel, immunoprecipitated Slx5). WBs were probed as in (C).

(F) The Slx5 middle domain (Slx5-Md) is required for interaction with Euc1. Y2H assays with AD-Euc181-183 and BD-Slx5 constructs. Slx5-Md: aa 201-335, Slx5-Md∆: ∆aa 201-338. Serial dilutions were spotted and cells were grown at 30°C for 4 days. See also Fig. EV3C.

(G) Euc1-SBM constructs show defective binding to the Slx5-Md. Y2H assay with the indicated constructs as in (D). See Appendix Fig. S6A for expression controls.

(H) Schematic representation of Euc1, Slx5 and Slx8, domain features and interactions. Domains and protein lengths are drawn to scale, numbers below each bar denote amino acid positions. The mapped interaction between Euc1 and Slx5 is indicated by an arrow. CC: coiled-coil domain, SBM: Slx5-binding mutant, S: SUMO, SIM: SUMO interacting motif.
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 6

Figure 6. Specific Euc1-Slx5 interactions are required for ub-hotspots and Euc1 ubiquitylation.

(A) Euc1 Slx5-binding mutations impair Euc1 ubiquitylation. His-ubiquitin-modified proteins were enriched by denaturing NiNTA-PDs as described in Fig. 4E. 3FLAG-tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter. NiNTA-PD eluates were probed with FLAG and ubiquitin (P4D1) antibodies. Whole cell lysates (input) were probed with FLAG and Dpm1 antibodies.

(B) The Slx5-SIMs and Slx5-Md are required for Euc1 ubiquitylation. NiNTA-PDs for His-ubiquitin as in (A). All strains expressed wild-type 3FLAGEuc1 from plasmids under the ADH promoter and His-ubiquitin. Slx5 constructs were expressed from plasmids under control of the endogenous promoter. WBs were probed as described in (A), Slx5-levels were probed using an HA antibody. Asterisk denotes a non-specific band. Slx5-SIM*: SIMs 1-4 were mutated as described (Xie et al, 2010), for SIM5, aa 477-479 (IIV) were mutated to alanines. To avoid possible mislocalization by deletion of the Slx5-Md, which overlaps with a putative NLS (Westerbeck et al, 2014), an N-terminal NLS was fused to all constructs. See Appendix Fig. S7C for HU complementation.

(C) Euc1 Slx5-binding mutations (SBM1/SBM2) reduce/abolish ub-HSs and recruitment of Slx5 to ub-HSs. ChIP-qPCR for ub-K48 (top) or Slx5 (using Slx5 antibody, bottom) in euc1∆ ubx5∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter. Data represent means ± SD (n = 3). See also Appendix Fig. S7E-G.

(D) Slx5-SIMs and the Slx5-Md are required for the formation of ub-HSs and recruitment of Slx5. ChIP-qPCR for ub-K48 (left) or Slx5 (anti-HA ChIP, right) in ubx5∆ cells or slx5∆ ubx5∆ cells complemented with plasmids expressing the indicated Slx5-constructs under control of the SLX5 promoter. Data represent means ± SD (n = 2). See also Appendix Fig. S7H.

(E) Schematic model for the proposed sequence of events at ub-hotspots. See main text (Discussion) for details. SIM: SUMO-interacting motif, Md: middle domain, S: SUMO, Ub: ubiquitin.
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 7

Figure 7. The ub-hotspot pathway is required to regulate aberrant Euc1 function.

(A) Deletion of EUC1 does not lead to ub-HS adjacent gene deregulation, but rather widespread transcriptome changes. Total RNA isolated from WT and euc1∆ cells was polyA-enriched and sequenced (RNAseq, triplicates). Significance testing was based on the Wald test, see Materials and Methods for details. See also Appendix Fig. S8A for quantification of selected transcripts by qPCR.

(B) RNAseq transcriptome analysis of EUC1 overexpression as in (A). ∆euc1 cells with pGAL-EUC1 or pEUC1-EUC1 (control) integrated at the URA3-locus (YIplac211) were grown to mid-log phase and 2% galactose was added for 3h. See also Appendix Fig. S8E.

(C) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol. For (C, D, F), serial dilutions of the indicated strains were spotted and grown on YPD control (or selective media) plates or under conditions as indicated. See also Fig. EV4A-B.

(D) EUC1 overexpression toxicity depends on DNA binding and transactivation. The indicated EUC1 alleles with endogenous or galactose-inducible promoters were integrated at the URA3 locus (YIplac211, euc1∆ background) and spotted on glucose control or galactose plates to induce EUC1 overexpression. See also Appendix Fig. S9A-C.

(E) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding. ChIP-qPCR of Euc1 after 3 hours galactose induction. Note that IP/input ratios of Euc1 signals are shown, also for the control locus (contr., TOS1 promoter) to highlight Euc1 binding at non-ub-HS sites. STI1-CIN5: intergenic region. TEC1-us: upstream (promoter) region. Data represent means ± SD (n = 3). See also Fig. EV4C-D.

(F) Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity. Experiment as in (D), but with concomitant plasmid-borne overexpression (ADH promoter) of SLX5 and SLX8 (bottom panels) and on media selecting for SLX5/SLX8 plasmids. See also Fig. EV4E and Appendix Fig. S9D.
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 8

Figure 8. EUC1 shows genetic interactions with regulators of gene expression upon thermo- and DMSO-stress.

(A-B) EUC1 displays negative genetic interactions with genes involved in general and specific transcriptional regulation (A) and in particular with members of the Rpd3L histone deacetylase complex (B). For (A-D), serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11, see Appendix Fig. S10C-F for details.

(C) Plasmid-borne EUC1 complements genetic interactions with Rpd3L subunits. Empty vector (-) or a plasmid encoding EUC1 with its endogenous promoter were transformed in the indicated strains and spotted on selective media.

(D) EUC1 and Rpd3S act in a common pathway which is redundant with Rpd3L. rco1∆ and eaf3∆ (Rpd3S) show similar phenotypes when paired with sds3∆ (Rpd3L) as euc1∆ and are epistatic with euc1∆.

(E) Graphic summary of EUC1 genetic interactions as tested in (A-D) and Appendix Fig. S10. Arrows indicate negative genetic interactions upon cold (blue), heat (red) or DMSO stress (black), dashed grey arrows indicate epistatic relationships.
Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance (2019)
Figure 9

Figure 9. Euc1-mediated ub-hotspots and Slx5/Slx8 are crucial for a functional stress response under impaired gene expression control.

(A) Euc1 is recruited to ub-HSs upon mild heat stress (37°C, 90 min). ChIP-qPCR experiments with strains and conditions as indicated. Data represent means ± SD (n = 4), see Appendix Fig. S11B for statistical analysis.

(B) The ability to form ub-hotspots is crucial for endogenous EUC1 function. The genetic interaction of EUC1 with SDS3 (Rpd3L complex) was rescued with plasmid-borne EUC1-alleles and serial dilutions were spotted on selective media. (-) denotes empty vector. See also Appendix Fig. S12A-B.

(C) SLX5 also shows negative genetic interactions with SDS3 upon heat stress. Serial dilutions of the indicated strains were spotted on YPD and incubated as indicated. See also Appendix Fig. S12C-D. Note that all strains used in (A-C) contained an extra copy of MED11, see Appendix Fig. S10C-F for details.

EXPANDED VIEW FIGURE LEGENDS
A selective ER-phagy exerts procollagen quality control via a CALNEXIN-FAM134B complex (2018)
Figure 1

Figure 1. PCs are autophagy substrates and accumulate in lysosomes.

(A, B) Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 µm.

(C) Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 µm. (A-C) The insets show higher magnification (A = x4.68; B = x6.76; C = x7.33) and single colour channels of the boxed area.

(D) Quantification of GFP (A,B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean +/- SEM), n = 18 cells (MEFs and Saos2); n=12 (RCS) from 3 independent experiments.

(E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area.

(H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows.

(I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001.

(J) WT and CRISPR IDUA Saos2 lysed and analysed by western blot. Data are representative of 3 independent experiments.
A selective ER-phagy exerts procollagen quality control via a CALNEXIN-FAM134B complex (2018)
Figure 2

Figure 2. Autophagy sequesters PC molecules in the ER.

(A, B) U2OS expressing (A) GFP-LC3 or (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93; B = x3.42) from the boxed region are shown on the right. Scale bar = 10 µm.

(C) Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabeled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 µm.

(D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue).

Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules.

(E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).
A selective ER-phagy exerts procollagen quality control via a CALNEXIN-FAM134B complex (2018)
Figure 3

Figure 3. FAM134B is required for autophagy recognition of PC1.

(A). Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of total number of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h. n=20 cells per condition; three independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ***P<0.0001.

(B) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1, fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). Scale bar = 10 µm. Insets show magnification of the boxed area. Bar graph (on the left) shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=12, 10, 12, 10, 7, 10 cells per genotype respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. ns ≥0.05, *** P<0.0001.
A selective ER-phagy exerts procollagen quality control via a CALNEXIN-FAM134B complex (2018)
Figure 4

Figure 4. PC1 accumulates intracellularly in cells lacking Fam134b.

(A) WT and CRISPR-Cas9 Fam134b knockout MEFs were treated as indicated, lysed and analysed by western blot with the indicated antibodies. Western blots are representative of 4 independent experiments.

(B) WT and CRISPR-Cas9 Fam134b MEFs were immunolabeled for PC1 (568, red), nuclei stained with Hoechst (blue) and analysed by scanning confocal microscopy. Scale bar = 10 µm.

(C) CRISPR FAM134B MEF mock, wild type FAM134B-HA or FAM134Blir-HA transfected were immunolabeled for PC1 (568, red), LAMP1 (488, green) and HA (647, violet) and analysed by scanning confocal microscopy. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), quantification of n=10 cells per treatment; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed. ns ≥0.05, *** P<0.0001.
A selective ER-phagy exerts procollagen quality control via a CALNEXIN-FAM134B complex (2018)
Figure 5

Figure 5. CANX is required for autophagic targeting of PC1.

(A) Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes, treated with 100 nM BafA1 for 9h. n=18 cells/treatment; 3 independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ** P<0.005, *** P<0.0001.

(B) WT and Canx-/- MEFs were untreated or treated with BafA1 (10 μM) for 6 h, lysed and analysed by western blot with indicated antibodies. Filamin and β-actin were used as loading control. Dashed line indicates that unnecessary lanes were removed. Western blot is representative of 3 independent experiments.

(C) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). CST was added where indicated. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=8, 8, 12, 8, 8, 8 cells respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. *** P<0.0001.
A selective ER-phagy exerts procollagen quality control via a CALNEXIN-FAM134B complex (2018)
Figure 6

Figure 6. PCs are the main substrates of CANX-FAM134B complex

(A, B) Volcano plot comparing protein fold changes between WT versus Fam134b-/- (A) and WT versus Canx-/- MEFs (B). Significantly regulated proteins are labelled in red (log2 fold change > 1, -log10 p>1.3). Red dots with blue ring indicate collagen 1 peptides. Graphs represent statistics from three separate experiments for each genotype.

(C) (left) Venn diagrams represent the number of identified peptides significantly enriched in Fam134b-/- and Canx-/- MEFs. (right). List of peptides upregulated in both Fam134b-/- and Canx-/- MEFs.