EMBO/SourceData huggingface dataset:
Figure 7(A) Representative image of vimentin+ fibroblasts (in green) expressing PC (identified using Arl13b, in red) in a normal colon. Nuclei were stained with DAPI (in blue). Most PC are detected on vimentin+ cells (arrow), and few on vimentin-negative epithelial cells (arrow head). (B,C) Quantitative analysis of PC presence on tumoral and peri-tumoral regions of colons from CRC patients (n=28) at tumor (T) stages 1 to 4. The panels represent quantification of PC expression (B) of vimentinpositive cells per mm2 and (C) per vimentinpositive cells as arbitrary unit (AU, see material and methods). Images were acquired on an Andor Dragonfly Spinning Disk Confocal microscope and analyzed with the Imaris software. Quantification of PC and Vimentin+ fibroblasts was done on at least 5 random fields (about 1mm2 each) per sample using ImageJ software.
Figure 1(A) Immunoblot analysis of caspase-1 and gasdermin D in WT or Nlrp3-/- unprimed and LPS-primed BMDMs left untreated [Medium alone (Med.)] or assessed 20 hr after stimulation with the supernatant (Sup.) of or assessed 20 hr after infected with WT C. perfringens (WT C. per), ΔpfoA C. perfringens (ΔpfoA), Δcpa C. perfringens (Δcpa, lacking lecithinase) or ΔpfoAΔcpa C. perfringens (ΔpfoAΔcpa) at MOI 100.(C) Immunoblot analysis of caspase-1, gasdermin D and GAPDH (loading control) in WT or mutant BMDMs left untreated or LPS-primed and assessed 3 hr after stimulation with lecithinase (Lec.).(D) Release of IL-1β (top), IL-18 (middle-top), death (middle-bottom) and TNF (bottom) of BMDMs after treatment as in C.
Figure 2(A) Concentration of IL-18 in the serum of WT mice, 8 hr after intraperitoneal infection with 4 × 108 colony-forming units (CFUs) of C. perfringens (WT C. per, n = 14), ΔpfoA C. perfringens (ΔpfoA, n = 13), Δcpa C. perfringens (Δcpa, n = 11) or ΔpfoAΔcpa C. perfringens (ΔpfoAΔcpa, n = 12). (B) Concentration of IL-18 in the peritoneal fluid of WT mice, 4 hr after intraperitoneal infection with 4 × 108 CFUs of C. perfringens (WT C. per, n = 14), ΔpfoA C. perfringens (ΔpfoA, n = 15), Δcpa C. perfringens (Δcpa, n = 17) or ΔpfoAΔcpa C. perfringens (ΔpfoAΔcpa, n = 14). (C) Concentration of IL-18 in the serum of WT mice (n = 10), Nlrp3-/- mice (n = 9), and Casp1/11-/- mice (n = 19), 6 hr after intraperitoneal infection with 1 × 108 CFUs of C. perfringens. (D) Concentration of IL-18 in the peritoneal fluid of WT mice (n = 10), Nlrp3-/- mice (n = 9), and Casp1/11-/- mice (n = 19), 3 hr after intraperitoneal infection as described in C.(G) Survival of WT (n = 19), Nlrp3-/- (n = 26) and Casp1-/- (n = 10) mice, after intraperitoneal administration of 0.312 Units/ml of lecithinase (Lec.).(J) Survival of WT mice administered with either PBS (n = 20) or MCC950 (n = 18), followed by intraperitoneal administration of 0.312 Units/ml of lecithinase (Lec.) as in H.
Figure 3(A) Confocal microscopy analysis of CD11b (green) and lecithinase (red) in unprimed WT BMDMs left untreated [Medium alone (Med.)] or assessed 1 hr after stimulation with Alexa Fluor 568 labelled lecithinase (AF568-Lec.) in the absence or presence of cytochalasin D (Cyto D; 50 μM) or methyl-β-cyclodextrin (MCD; 5 mM).(D) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 3 hr after stimulation with perfringolysin O (PFO), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of methyl-β-cyclodextrin (MCD; 5 mM). (E) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in D. (F) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 4 hr after infection of with S. Typhimurium (S. Tm.; MOI, 5), or 30 min after stimulation with nigericin in the absence or presence of cytochalasin D (Cyto D; 50 μM). (G) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in F. (H) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 4 hr after infection of with S. Typhimurium (S. Tm.; MOI, 5), or 30 min after stimulation with nigericin in the absence or presence of latrunculin B (Lat B; 1 μg/ml). (I) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in H.
Figure 4(B) Confocal microscopy analysis of LAMP1 (green) and lecithinase (red) in WT BMDMs left untreated or assessed 1 hr after stimulation with AF568-lecithinase. (C) Three-dimensional surface rendering of LAMP1 (green) and lecithinase (red) shown in B. (D) Line scan values for LAMP1, AF568-lecithinase, and DAPI taken from the white dashed line in the gold-colored inset in B. These values were plotted and fitted with a single Gaussian equation (Arbitrary Unit (AU)).
Figure 5(A) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated [Medium alone (Med.)] or assessed 3 hr after stimulation with lecithinase (Lec.), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of bafilomycin A (Baf A; 100nm). (C) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 16 hr after infection with F. novicida (F. novi., MOI 100), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of ammonium chloride (NH4Cl; 20 μM).(B) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in A. (D) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in C.(F) Flow cytometry analysis of WT BMDMs stained with acridine orange (1μg/ml) and pre-treated with various inhibitors (MCD, Cytochalasin D, Bafilomycin A, ammonium chloride) for 30 min, and assessed 60 min after stimulation with lecithinase (Lec.). WT BMDMs stained with acridine orange were also assessed 60 min after stimulation with heat-treated lecithinase (Heat).
Figure 6(E and F) IncuCyte live-imaging analysis of the viability of BMDMs after treatment as in A.(G and H) IncuCyte live-imaging analysis of the viability of BMDMs after treatment as in C.(I) Release of IL-1β (top), IL-18 (middle) and death (bottom) of WT or Ninj1-/- BMDMs left untreated or LPS-primed and assessed for a range of time points after stimulation with lecithinase (Lec.) or 16 hr after transfection with LPS (LPS trans.).(J) Immunoblot analysis of caspase-1, gasdermin D and NINJ1 of WT or Ninj1-/- BMDMs left untreated or LPS-primed and assessed 3 hr after stimulation with lecithinase (Lec.) or 16 hr after transfection with LPS (LPS trans.).
Figure 1(B) Chlamydomonas reinhardtii (Cr) UBA5 and UFC1 are active E1 and E2 enzymes. SDS-PAGE analysis showing transfer of UFM1 to CrUBA5 and CrUFC1. The gels are run in non-reducing conditions except where otherwise specified. The presented gel is representative of two independent experiments. BME: β-mercaptoethanol; ATP: Adenosine triphosphate.(C) RPL26 mono- and di-UFMylation is lost in Chlamydomonas reinhardtii (Cr) uba5 and ufl1 mutants. Liquid TAP cultures were either left untreated (control) or treated for 24 hours with 200 ng/mL tunicamycin. Protein extracts were analyzed by immunoblotting with anti-UFM1 antibodies. Total proteins were analyzed by Ponceau S staining. 12 hours and 24 hours treatment replicates are shown in Fig. S3C. Right Panel, Quantification of UFMylated RPL26. RPL26 mono-UFMylated; RPL26-(UFM1)2: RPL26 di-UFMylated.(D) Chlamydomonas reinhardtii (Cr) UFMylation pathway mutants are sensitive to ER stress triggered by tunicamycin. Liquid TAP cultures of wild type (wt), uba5, ufl1 and ire1 mutants were either left untreated (control) or treated for 3 days with 200 ng/mL of tunicamycin. Left panel, representative images of control and treated liquid cultures taken 3-days after incubation. Middle Panel, optical density (OD) 600 (OD600) quantification of each genetic background under control conditions. Bars represent the mean (± SD) of 5 biological replicates. Two-tailed unpaired t-tests were performed to analyze the differences between wild type and mutants. Right Panel, normalized OD600 quantification of each genetic background under tunicamycin treatment conditions. Bars represent the mean (± SD) of 5 biological replicates. Two-tailed unpaired t-tests were performed to analyze the differences between wild type and mutants. ns, p-value > 0.05; ***, p-value < 0.001. BR: Biological Replicate.
Figure 2(F, G) C53 IDR is essential for UFM1 interaction. HsC53 (B) and AtC53 (C) IDRs are necessary to mediate the interaction with AtUFM1 and HsUFM1 respectively. MBP-AtC53ΔIDR: MBP-AtC53(1-239, (KGSGSTSGSG)2, 373-549); MBP-HsC53ΔIDR: HsC53(1-262, (KGSGSTSGSG), 317-506). F, G) Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies.(H) AtC53sAIM cannot interact with AtUFM1. Individual or combinatorial mutations in sAIM1 (1A: W276A), sAIM2 (2A: W287A) and sAIM3 (3A: W335A) suggest sAIM1 is crucial for UFM1 interaction.
Figure 3(B) The cAIM peptide cannot outcompete HsUFM1-HsC53 interaction. Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies. cAIM peptide was used to a final concentration of 200 µM. HsC53sAIM: HsC53W269A, W294A, W312A.(C, D) Microscopy-based protein-protein interaction assays showing unlike GABARAP-C53 interaction, UFM1-C53 interaction is insensitive to cAIM peptide competition. Glutathione-sepharose beads were prepared by incubating them with GST-GABARAP (C) or GST-HsUFM1 (D). The pre-assembled beads were then washed and mixed with 1 µM of HsC53 containing increasing concentrations of cAIM peptide (0-100 µM). The beads were then imaged using a confocal microscope. Left Panel, representative confocal images (inverted grayscale) for each condition are shown. Right panel, normalized fluorescence is shown for each condition with the mean (± SD) of 4 replicates. Unpaired two-samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and wild type with 100 µM AIM peptide. ns, not significant, p-value > 0.05, ***, p-value < 0.001.
Figure 4(B) Binding of AtUFM1 to AtC53 IDR leads to a general drop in signal intensity. NMR signal intensity of AtC53 IDR (100 µM) in the presence of 300 µM AtUFM1 normalized to signal intensity of AtC53 IDR alone. Bars corresponding to residues in the AIMs are highlighted.(D) The backbone amide signals of AtC53 IDR shift upon AtUFM1 addition in a concentration-dependent manner. Insets of overlaid 1H-15N HSQC spectra of isotope-labelled AtC53 IDR (100 µM) showing chemical shift perturbations of individual peaks from backbone amides of AIM residues in their free (gray) or bound state to unlabelled AtUFM1. Chemical shifts are indicated with arrows.
Figure 5(B, Conversion of sAIM into cAIM leads to reduced UFM1 binding and stronger ATG8 interaction. Bacterial lysates containing recombinant proteins were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies. AtC53sAIM: AtC53 (W276A, W287A, W335A); AtC53cAIM: AtC53(IDWD274WDDI, IDWD285WDDI, IDWD333WDDI); ATG8LDS: ATG8YL50AA.(G) AtC53cAIM forms more GFP-ATG8A colocalizing puncta upon ER stress. Upper Panel, representative confocal images of transgenic Arabidopsis seedlings co-expressing C53-mCherry (magenta), C53sAIM-mCherry and C53cAIM-mCherry with GFP-ATG8a in c53 mutant background under normal condition and after tunicamycin stress. 6-day old seedlings were incubated in liquid 1/2 MS medium with 1% sucrose supplemented with DMSO as control or tunicamycin (10 μg/ml) for 6 hours before imaging. Scale bars, 30 μm. Inset scale bars, 10 μm. Right Panel, Quantification of the C53-autophagosomes (C53-APG) per normalized Z-stacks. Bars represent the mean (± SD) of at least twenty roots from 3 biological replicates for each genotype and treatment. Unpaired two-samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and mutants. ***, p-value < 0.001.
Figure 3(A) Representative western blot of proteins pulled down by IP against Pum1 compared to IgG from wild-type mice brain without (left) and with (right) RNase treatment. In this panel, after IP-Pum1, we immunoblotted for Pum1 (positive control), Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10(B) Representative western blots with and without RNase. Molecular weights at the right are in kilodaltons (kDa). IP against IgG was used as a negative control, and Input (1% from the initial protein lysate as a loading control.
Figure 4(A-C) Representative western blots of Pum1, Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10 in (A) cerebellum, (B) hippocampus, and (C) cortex in both male (left panel) and female (right panel) WT, Pum1+/-, and Pum1-/- mice. All the experiments were conducted with equal numbers of 10-week-old male and female mice per genotype, for a total of at least 6 mice per genotype Graphs below show quantification for each protein by brain region, sex, and genotype. All data were normalized to Gapdh protein levels. P values were calculated by two-tailed Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001. P1 indicates Pum1.(D) Heatmap showing 166 microRNAs from cerebella of Pum1-/- male and female mice that were dysregulated (fold change -3 to +3) relative to wild-type cerebellum. (E) Heatmap showing mRNA quantification by qPCR for 49 targets co-bound by a minimum of eight dysregulated miRNAs (>25% change) from panel D. Statistical significance and magnitude of dysregulation are illustrated for both male and female
Figure 5(C) GSEA analysis scores of Pum1, Pum2, Rbfox3, and Ago2 HITS-CLIP data plotted against Fmrp HITS-CLIP data divided into 10-percentile ranked bins shows the shared targets are among the top percentiles of targets for each protein.
Figure 6(A, B) IP against PUM1 from Subject 1 (S1, R1147W) (A) and Subject 2 (S2, T1035S) (B) compared to their respective age-, sex-, and cell type- matched healthy controls confirm the interactions between PUM1 (used here as a positive control), and PUM2, FMRP, AGO2, CNOT1, and MOV10. Input (1%) was used as a loading control and IP against IgG was used as a negative control. GAPDH was used here as loading control, see Methods for quantification.(C, D) Representative western blots (C) and relative quantification (D) of protein levels for PUM1, PUM2, FMRP, AGO2, CNOT1, and MOV10 in PADDAS patient-derived and PRCA patient-derived cells compared to their respective controls. Data were normalized to GAPDH protein levels.
Figure 7(B) Representative western blots (left panel) and relative quantifications (right panel) of PUM1 and its interactors (AGO2, CNOT1, FMRP, and MOV10) in R1147W patient-derived cell lines after Myc-PUM1-WT expression.(C) mRNA quantification of the top 15 shared target genes from panel A in R1147W patient-derived cell lines after Myc-PUM1-WT expression.
Figure 2HIV-1YU-2 was preincubated with the indicated concentrations of antibodies or BiCEs and subsequently incubated with HIV reporter TZM-bl cells. 48 h later, HIV-infection was measured as luciferase activity quantified by luminescence. For each sample, a normalized HIV-infection is calculated by subtracting RLU of control (no virus added), and then normalized to virus control (no antibody or BiCE added) set to 100%. n = 3 independent experiments (10-1074 and 3BNC117) or n = 4 independent experiments (scFv3BNC117-Nb75) or n = 7 independent experiments (scFv10-1074-Nb75) or n = 1 experiment (scFv10-1074, Nb75 and anti-tetanus IgG), each experiment showing an average of triplicate wells. MW = 150 kDa (10-1074, 3BNC117 and anti-tetanus IgG), MW = 43 kDa (scFv10-1074-Nb75), MW = 45 kDa (scFv3BNC117-Nb75), MW = 28 kDa (scFv10-1074) and MW = 15 kDa (Nb75).Raji cells expressing HIV-1YU-2 envelope (Raji-Env) or Raji control cells (Raji-Ctr) were incubated with 10-1074 or scFv10-1074 or with controls (only secondary antibody or streptavidin added). Binding of 10-1074 and scFv10-1074 was assessed by flow cytometry using a BV421-conjugated anti-IgG Fc antibody or streptavidin, respectively. The numbers indicate the % of cells with bound antibody. One experiment is shown.
Figure 3C3 deposition by the indicated antibodies or BiCEs. Results are expressed as the C3 MFI % fold change compared to no antibody treatment. n = 3 donor serum. Data information: All data are presented as mean ± SEM.CDC triggered by the indicated antibodies or BiCEs. CDC was calculated as the relative % of dead cells compared to no antibody treatment. n = 3 donor serum. Data information: All data are presented as mean ± SEM.
Figure 4C3 deposition by the indicated antibodies or BiCEs. Results are expressed as the C3 MFI % fold change compared to no antibody treatment. n = 6 donors (10-1074, 3BNC117, 10-1074+3BNC17 and scFv10-1074-Nb75+scFv3BNC117-Nb75) or n = 8 donors (no antibody treatment, scFv10-1074-Nb75 and scFv3BNC117-Nb75) or n = 2 donors (scFv10-1074, Nb75 and anti-tetanus IgG). Data information: Data are presented as median with interquartile range. Significance was determined by comparing each antibody or BiCE to no antibody treatment. Only significant differences are shown; *P < 0.05 and **P < 0.01, Wilcoxon signed-rank test. When n ≤ 2, no statistical analysis was performed.Primary CD4 T cells infected with HIV-1NL4-3-eGFP and uninfected CD4 T cells were incubated with NHS and the indicated antibodies or BiCEs in presence of an HIV-inhibitor. The frequency of eGFP+ cells was analyzed by flow cytometry at the indicated time points. The numbers indicate the % of eGFP+ cells. One representative donor is shown.Induction of eGFP+ elimination over time by the indicated antibodies or BiCEs. Results are expressed as the relative % of eGFP+ cells compared to no antibody treatment. n = 6 donors (10-1074, 3BNC117, 10-1074+3BNC17 and scFv10-1074-Nb75+scFv3BNC117-Nb75) or n = 8 donors (no antibody treatment, scFv10-1074-Nb75 and scFv3BNC117-Nb75) or n = 2 donors (scFv10-1074, Nb75 and anti-tetanus IgG). Data information: Data are presented as median with interquartile range. Significance was determined by comparing each antibody or BiCE to no antibody treatment. Only significant differences are shown; *P < 0.05 and **P < 0.01, Wilcoxon signed-rank test. When n ≤ 2, no statistical analysis was performed.
Figure 1Viability curve of cells following 24h of recombinant Pol II expression induction by removal of tetracycline (Tet-Off system) and treatment with α-amanitin (black arrow-head). Untransfected Raji (WT: black) is shown for reference. For all experiments presented in this article, cells are collected after 24 hours of α-amanitin treatment for CTD-∆5 and YFFF mutants expressing cells since CTD-∆5 is unstable after 48h (see panel C). Error bars representing top and bottom values are based on the average of 3 independent experiments.Western blot analysis of the recombinant RPB1 expression (rWT, YFFF, and ∆5) assessed by (HA)-tag expression after 24 or 48 hours of α-amanitin treatment following 24h of induction. Untransfected Raji extracts are shown for comparison (WT). Total RPB1 levels are represented by the F12 western blot. Tubulin was used as a loading control.Volcano plot of differentially expressed genes in chromatin associated RNA-seq (ChrRNA) data sets: protein coding genes UP (purple) and DOWN (pink) regulated in B relative to A after 24h hours α-amanitin treatment (n=22810). Experiments were done in biological duplicates. Threshold: log2 fold change ≥ 3, p-value < 0.05.
Figure 2Example of ChrRNA read-through phenotype at the 5' (anti-sense) and 3' (sense) ends in CTD-∆5 at the GBE1 locus.Boxplots of ChrRNA read-through (RT) indexes based on signal over 20kb upstream (anti-sense) and 20kb downstream (sense) regions of coding genes in rWT (dotted grey line) and CTD-∆5 mutant (blue). Units are asinh transformed. Experiments were done in biological duplicates. Boxplots represent minimal and maximal values, first and third quartiles with median value as central band. P-values were calculated using two-sided Wilcoxon tests.
Figure 3Example of Pol-HA ChIP-seq showing global spreading of Pol II along the genome accompanied by a loss of Pol-II accumulation on coding genes in CTD-∆5 (orange) compared to rWT (black) and their associated ChrRNA-signal.Average metagene profiles comparing Pol-II, H3K4me3 and H3K4me1 signals on active promoters (top) and active enhancers (bottom) in rWT and CTD-∆5. Experiments were done in biological duplicates however only the sample with the best signal to noise ratio was used for subsequent analysis. P-values were calculated using two-sided Wilcoxon tests.Example of Pol-HA, H3K4me3 and H3K4me1 Chip-seq signals in rWT and CTD-∆5 illustrating the metagene profiles in figure 3B. Top: Light green rectangles highlight putative active enhancers in rWT.
Figure 4Example showing ChrRNA-seq (top) and its associated PolyA RNA-seq signals (bottom) in rWT and CTD-∆5.Boxplots of PolyA read-through (RT) indexes based on signal over 20kb upstream (anti-sense) and 20kb downstream (sense) regions of coding genes in rWT (dotted grey line) and CTD-∆5 mutant (blue). Units are asinh transformed. Experiments were done in biological duplicates. Boxplots represent minimal and maximal values, first and third quartiles with median value as central band. P-values were calculated using two-sided Wilcoxon tests.
Figure 5Volcano plot comparing the Pol II interactome in rWT and CTD-∆5 mutant. Left: Proteins lost in CTD-∆5, Right: Proteins enriched in CTD-∆5. Highlighted are the subunits of the Mediator (purple) and Integrator complexes (orange) CTD phosphatases scaffolds (pink), Ubiquitin ligases (light blue) and SOSS complex subunits (red). In black are highlighted some chosen interactors gained by the CTD-∆5 Threshold: log2 fold change ≥ 4; p-value < 0.05. Data based on three independent biological replicates. The table in the figure sums up the proteins that do not change significantly; dotted lines connected to the table represent the region where these proteins are found in the volcano
Figure 6Example of CTD-∆5 polymerases transcribing through TAD borders marked by CTCF sites (highlighted in light purple). HiC map on top represents the rWT interactions shown in the zoom in panel A. Purple boxes represent TADs called in human B cells CTCF ChIP-seq in rWT and CTD-∆5 are both shown in black and their associated ChrRNA-seq signal are also shown.
Figure 1(A) Anti-NANP5 and anti-C-CSP serum IgG. Circles represent ELISA area under the curve (AUC) values for individual donors (n=12). (B) Ten most frequent Ig heavy and light chain V gene pairs in PfCSP-reactive memory B cells (MBCs; n=1172).somatic hypermutation (SHM) count (E) in IGHV3-21 and IGHV3-33 genes from MBCs(F) NANP10 (left) and C-CSP (right) binding of FL-CSP-reactive mAbs (n=177). Circles represent ELISA AUC values for each mAb.
Figure 2(C) VH SHM load of VH3-21 and non-VH3-21 mAbs. mAbs with unmutated VH are highlighted in green.(D) Amino acid (aa) VH replacement (red bars) and silent (black bars) SHM in VH3-21 mAbs (n=113). FWR, framework region; CDR, complementarity-determining region.
Figure 3(D) C-CSP reactivity of the α-TSR-specific mAb 1710 in a blocking ELISA with C-CSP-specific VH3-21 (n=21; upper panel) or non-VH3-21 (n=17; lower panel) mAbs with the indicated SPR affinities. mAb 1710 binding without blocking (blue) and after self-blocking (gold) is shown for comparison.(E) C-CSP reactivity of mAb 1512 in a blocking ELISA with mAbs (with the indicated SPR affinities) that do not block C-CSP binding of mAb 1710 (open circles; D). mAb 1512 binding without blocking (purple) and after self-blocking (magenta) is shown for comparison.(F) ELISA cross-reactivity with Pf-7G8 FL-CSP of CSP-specific mAbs that block (left) or do not block (right) mAb 1710 binding to NF54 C-CSP.