EMBO/SourceData huggingface dataset:
Figure 2 A. M.HpaII was crosslinked into a plasmid to generate p4xDPC and then incubated in nucleoplasmic egg extracts (NPE) in the presence or absence of 50 μM of SUMOi. DPC pull-down under stringent conditions was performed at the indicated time points and the recovered samples were immunoblotted for crosslinked M.HpaII (Larsen et al, 2019). Note that p4xDPC, which contains four M.HpaII DPCs, is sometimes used to increase sensitivity towards modified M.HpaII species. Identical results were obtained with plasmids containing one or four DPCs.  B. p4xDPC or p4xDPCSUMO (generated by crosslinking SUMO∆GG-M.HpaII) were incubated in NPE and DPC pull-down performed as in (A). Note that priming M.HpaII with SUMO stimulates rapid poly-SUMOylation of the DPC in NPE. Thus, this substrate was frequently used in subsequent experiments to stimulate DPC poly-SUMOylation.  C. p4xDPCSUMO was incubated in NPE and plasmids analyzed as in (A). Following plasmid pull-down, the samples were split and either left untreated or treated with the SUMO protease Ulp1.  I. Representative images of U2OS cells transfected with Myc-PIAS4 expression plasmid that were left untreated or exposed to 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and Myc antibodies. Scale bar, 5 μm.  J. HeLa or HeLa/GFP-DNMT1 cells left untreated or exposed to 5-azadC for 30 min were lysed and subjected to GFP IP under stringent conditions. After extensive washing, individual IPs were incubated with an equal amount (800 μg) of whole cell lysate of HeLa cells transfected with Myc-PIAS4 expression construct (Fig. EV2J), washed and immunoblotted with antibodies to Myc, SUMO2/3 and GFP. *, cross-reactive band.  K. HeLa/GFP-DNMT1 cells transfected with previously validated siRNAs targeting established SUMO E3 ligases (Fig. EV2L and Methods section) were treated with 5-azadC for 30 min, collected and subjected to GFP immunoprecipitation under denaturing conditions, and immunoblotted with antibodies to SUMO1, SUMO2/3, ubiquitin and GFP.
Figure 3 B. p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [α-32P]dATP. Where indicated, 50 μM of SUMOi was added to the extracts. Reaction samples were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled. Red arrowheads indicate OC molecules that have not yet undergone repair. C. Samples in (B) were recovered by stringent DPC pull-down and immunoblotted for crosslinked M.HpaII.  J. As in (I), but using RNF4-depleted CSF-arrested extracts reconstituted with recombinant RNF4 WT or C130A from (H).  L. p4xDPCSUMO was subjected to sequential addition of NPE and CSF-arrested extract as in (I). CSF-arrested extracts from (K) were supplemented with recombinant RNF4, and the DPC plasmid was recovered by DPC pull-down at the indicated time points and immunoblotted against M.HpaII.
Figure 4 D. HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse-labeled with 5-azadC in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.  E. HeLa cells transfected with indicated siRNAs were treated with 5-azadC and/or SUMOi in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.  F. HeLa cells transfected with indicated siRNAs were pulse-labeled or not with 5-azadC for 30 min in late S phase according to the experimental setup in (A). Following 5-azadC withdrawal cells were incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies.  H. Immunoblot analysis of siRNA-treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later.
Figure 5 B. Representative time-lapse sequences of mitotic progression in cells in (A). NEBD corresponds to t=0. White arrowheads indicate cells undergoing division. Scale bar, 10 μm.  E. HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block. Six h after release from the block, cells were pulse-labeled with 5-azadC for 30 min. Mitotic cells were isolated by shake-off 8 h later, washed and incubated or not with MPS1 inhibitor (MPS1i) and collected at the indicated times. Cells were then processed for immunoblotting with indicated antibodies.
Figure 1(A-B) Spinal cord sections from 5-month-old WT and Tmem106b-/- mice were stained with anti- neurofascin, anti- LAMP1 and anti-Cath D antibodies. Accumulation of LAMP1 and CathD positive vacuoles at the distal end of AIS was observed in Tmem106b-/- motor neurons. Representative images from three independent mice were shown. Scale bar = 10 µm. n=3. Data presented as mean ± SEM. Unpaired Student's t test. ****, p<0.0001.(C-F) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from brain (C and E) and spinal cord (D and F) of 16-month-old WT and Tmem106b-/- mice. n=5. Data presented as mean ± SEM. Unpaired Student's t test. *, p<0.05, ***, p<0.001, ****, p<0.0001.
Figure 2(A) Tmem106b-/-Grn-/- mice do not show any changes in body weight. Body weight of 5-month-old mice of indicated genotypes were measured and analyzed. n=6-10.(B Tmem106b-/-Grn-/- mice show reduced activities in the open field test. 4.5-month-old mice were used in the study.C) Tmem106b-/-Grn-/- mice show reduced activities in the open field test. 4.5-month-old mice were used in the study. Total movement was quantified. n=8-10. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ****, p<0.0001.(D Tmem106b-/-Grn-/- mice show abnormal hindlimb clasping behavior. 5-month-old mice were used in the study.E) Tmem106b-/-Grn-/- mice show abnormal hindlimb clasping behavior. 5-month-old mice were used in the study. Severity score was quantified. n= 8-10. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.
Figure 5(A, B) Retinal sections from 5-month-old mice were stained with indicated antibodies. Thickness of each layer, number of Brn3a positive cells (retinal ganglion cells) per section and length of outer segment as indicated by opsin staining are quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.(C Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.D) Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified.(E Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.F) Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control.(G, H) Increase in microglia activation in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by IBA1 staining. The number of IBA1 positive cells per section was quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.
Figure 6(A Heatmap illustrating gene expression changes in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- cortex samples isolated from 2.7-month-old mice. n=3. Genes with FDR p≤0.05, LogFC≥0.5 or ≤0.5 between WT and Tmem106b-/-Grn-/- groups are plotted based on hierarchical clustering analysis. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.B) Heatmap illustrating gene expression changes in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- spinal cord samples isolated from 2.7-month-old mice. n=3. Genes with FDR p≤0.05, LogFC≥0.5 or ≤0.5 between WT and Tmem106b-/-Grn-/- groups are plotted based on hierarchical clustering analysis. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.(C) Heatmap illustrating gene expression changes in the inflammation and lysosome pathways in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- spinal cord samples. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.
Figure 7Western blot analysis of Galectin-3 and GPNMB in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.Western blot analysis of Galectin-3 and GPNMB in the brain lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.(E) Immunostaining of Galectin-3, CathD and CD68 in spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 10 µm.
Figure 8(A-B) Lipofuscin accumulation in the retina: the number of auto fluorescent puncta per section was quantified. ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. n=3-6. Scale bar = 50 µm.TMEM106B and PGRN deletion results in increased autofluorescence in the spinal cord of 5-month-old mice. n=3. Scale bar = 10 µm.TMEM106B and PGRN deletion results in increased autofluorescence in the thalamus of 5-month-old mice. n=3. Scale bar = 10 µm.(G, H) Western blot analysis of lysosomal proteins in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Asterisk indicates non-specific bands. n=3.
Figure 10(A Immunostaining of GFAP with LAMP1 and Cath D in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 5 µm.B) Immunostaining of IBA1 with LAMP1 and Cath D in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 5 µm.
Figure 11(A) Accumulation of autophagosomes and lysosomes in the spinal cord sections from a 5-month-old Tmem106b-/-Grn-/- mouse as shown by transmission electron microscope analysis. Scale bar = 1µm. Arrows: autophagosome; arrow heads: lysosomes; (a): axon.(B) Accumulation of myelin debris and amorphous inclusions in the microglia in the spinal cord sections from a 5-month-old Tmem106b-/-Grn-/- mouse as shown by transmission electron microscope analysis. Scale bar = 1µm. Arrows: myelin debris; arrow heads: dark amorphous inclusions; (m): microglia.(C) Western blot analysis of LC3-I and LC3-II in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ****, p<0.0001.(D Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice.E) Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Nuclear signals of TFE3 in IBA1 positive microglia were quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.
Figure 12(A, B) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from spinal cord (C5-C8) and brain of 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Spinal cord: n=3; brain: n=4. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05; **, p<0.01***, p<0.001, ****, p<0.0001.(C, D) Immunostaining of p62 and ubiquitin in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. The number of p62 or Ub positive puncta was quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.
Figure 1A. H3122 cells expressing endogenous EML4-ALK V1 and HEK293 transfected with YFP-EML4-ALK V1 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B. Violin plots showing the number of EML4-ALK V1 cytoplasmic foci per cell. Data represents 30-50 counts from three biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V1 KD by one-way ANOVA analysis. C. H2228 cells expressing endogenous EML4-ALK V3 and HEK293 transfected with YFP-EML4-ALK V3 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. D. Violin plots showing the number of EML4-ALK V3 cytoplasmic foci per cell. Data represents 30-50 counts from four biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V3 KD by one-way ANOVA analysis. H. Whisker plot showing the calculated velocity of single events in the kymograph. Data represent 20 counts from 10 kymographs. n=2. Error bar represents SD of two biological replicates. *P< 0.05 in comparison to YFP EML4-ALK V3 KD by unpaired t test.
Figure 2A, B. H3122 and H2228 cells were stained for anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.C. HEK293 cells transfected with YFP-EM4L-ALK V1 WT or V3 WT for 48 hrs and treated with 5% or 10% 1,6-hexanediol. Representative still images after 5 min (taken from time-lapse movies) are shown of cells treated with 10% 1,6-Hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci. D. Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5-10 counts from 3 independent experiments (n=3). Error bar represents SD of three biological replicates. ****P< 0.0001 in comparison to YFP-EML4-ALK V3 WT DMSO by one-way ANOVA analysis. E. Representative western blots of HEK293 transfected EML4-ALK V1 WT and V3 WT for 48 hrs. Cells were treated with either 5% or 10% 1,6-Hexanediol for 5 minutes. DMSO was used a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n=2 experiments.
Figure 3A, B, C, D. HEK293 cells were transfected with EML4-ALK V1 WT or V3 WT or Kinase Dead (KD) for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.
Figure 4B. HEK293 cells were transfected with EML4-ALK V1 deletion 12N blade for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.
Figure 5D. HEK293 cells were transfected with EML4-ALK V3 ∆JM 1058-1100 for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. E. Violin plot representing the number of cytoplasmic foci counted per cell from D. Data represent counts from at least 30 cells, n=3. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.
Figure 7B. HEK293 cells transfected with YFP-EML4-GyrB and either untreated (DMSO) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C. Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP-EML4-GyrB. Data represent counts from >30 cells, n= 4. ****P< 0.0001 in comparison to DMSO by unpaired t test.
Figure 1(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested. The ferritin content was determined by ELISA. Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin. The data are presented as the standard deviation from three different experiments.(B) Cells treated as in (A) were incubated in the presence of 1 μg/ml EGF for 2 h. Cells were fixed and processed for immunofluorescence using mouse anti‐EGF receptor and Alexa 594 conjugated goat anti‐mouse IgG. Chloroquine and leupeptin effectively inhibit degradation of EGF receptor.
Figure 2(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 or 10 μM lactacystin in the presence of Ponasterone A for 10 h and harvested. The ferritin content was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) Samples treated as in (A) were immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ferritin or ubiquitin by Western blot analysis. The arrows indicate the migration of H and L chains.(C) Cells were incubated with 1.0 × 10−7 M Tf(59Fe)2 for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 in the presence of Ponasterone A for 10 h and harvested and the amount of 59Fe in immunoprecipitatedferritin was determined.(D) Cells were treated as described in (A) and cell extracts were applied to size exclusion chromatography and the ferritin content, in selected fractions, was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(E) Cells were treated as described in (C), ferritin was then immunoprecipitated, eluted using 100 mM glycine, pH 2.5 and measured by ELISA. The amount of ferritin‐associated 59Fe was measured and the specific activity of ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.(F) Samples were treated as in (D), and ferritin was immunoprecipitated and analyzed by Western blot analysis using an antibody to ubiquitin.
Figure 3(A) FM3A (black) and ts85 (grey) cells were transiently transfected with a plasmid containing CMV‐regulated Fpn‐GFP and incubated in the presence of FAC (10 μM Fe) and 0.5 μM hepcidin for 24 h. Hepcidin was either maintained (+) or removed (−) to allow Fpn‐GFP localization at the plasma membrane. Cells were maintained at the permissive temperature (33°C) or moved to the restrictive temperature (39°C) and incubated for 6 h. Cells were harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) FM3A and ts85 cells were treated as in (A) but FAC was replaced with 1.0 × 10−7 M Tf(59Fe)2. Ferritin was immunoprecipitated and the specific activity of 59Fe‐ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.(C) ts85 cells were treated as in (B) and cell extracts applied to size exclusion chromatography. Ferritin content in selected fractions was measured by ELISA. Black bars represent assembled ferritin (>400 kDa), gray bars represent monomeric (50 kDa).(D) FM3A and ts85 cells were treated as in (A) but cells that had been incubated at the restrictive temperature (39°C) were then returned to the permissive temperature (33°C) and incubated in the presence of cycloheximide (75 μg/ml) and 10 μM FAC for 1 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.
Figure 4(A) Cells were then incubated at the restrictive temperature (39°C) for 6 h in the presence or absence of 100 μM DFO, with or without 100 μM chloroquine or 10 μM MG132. Cells were then harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) Cells treated as in (A) were incubated in the presence or absence of 5 mM 3‐methyladenine.
Figure 5(A) Δccc1/pGAL‐H+L cells were transformed with a plasmid containing a methionine regulated CCC1 (pMET3CCC1). Cells were grown in medium with galactose for 20 h, washed and incubated in galactose (•), glucose with 10 × methionine (▵), glucose (○) or glucose without methionine (▾) for 10 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate. The absence of methionine leads to expression of the vacuolar iron transporter Ccc1p.(B) erg6‐2 and erg6‐2 pGAL‐H+L strains were grown in medium with galactose and 250 μM FeSO4 for 24 h. Cells were then washed and incubated in galactose or glucose in the absence or presence of 50 μM MG132 for 7 h.(C) Cells were then harvested, ferritin levels determined by ELISA and immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ubiquitin by Western analysis. Cells with the erg6‐2 allele permit the entry of MG132 ( Lee and Goldberg, 1996).
Figure 1(A) Mitochondrial extracts from 143B cells, COX1∆ and COX2∆ mutant cybrids and their respective isogenic controls (CON-1 and CON-2) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The graphs (right) show densitometric quantifications of the signals normalized to CII subunit SDHA. The mean of three independent controls (CON) was set to 100 and all measurements were referenced to that value. The values represent mean ± SD (n=3-8). Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; β, p<0.01; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05; ∗∗p<0.01.(B) Digitonized mitochondria (digitonin-to-protein ratio of 2:1) were analyzed by BN-PAGE, followed by CI in gel activity (IGA) assays and Coomassie staining. The SC I+III2+IV1 band (b1) was excised from the control (143B) lane and the SC I+III2 bands (b2 to b4) were excised from the COX1∆ and COX2∆ lanes. Their protein compositions were subsequently analyzed by nano-LC/ESI-MS (see also Figure EV2). The relative position of the molecular weight marker is indicated on the left.(C) BN-PAGE was followed by CI-IGA assay and immunoblotting with the indicated antibodies (in brackets). The identity of the MRC complexes and SCs is: I+III2+IV1, SC containing CI, CIII2, and CIV; I+III2, SC containing CI and CIII2; I+III2plus, SC containing CI, CIII2 and a COX1-submodule; III2+IV, SC containing CIII2 and CIV; I, complex I; III2, CIII dimer; IV2, CIV dimer; subCOX1, COX1-containing subcomplexes.