EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Axotomised DIV 14-16 rat cortical neurons expressing p110δ and GFP or GFP alone. Axons were cut >1000 μm from the soma imaged for 14 h. Black arrows mark the cut site, white arrows marks the axon tip. See also Movies EV7 and EV8. (B) Percentage of regenerating axons 14 h after laser axotomy. Numbers on bars are injured axons per group, from 4 experiments. (C) Quantification of axon regeneration length 14 h post-axotomy. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments. (D) Quantification of time to start of regeneration. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments. (E) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone. Black arrow marks the cut site, white arrows mark the axon tip. (F) Percentage of regenerating axons of human hESC neurons, Numbers on bars are injured axons per group, from 5 experiments. (G) Quantification of axon regeneration length. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test. (H) Quantification of time to start of regeneration. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test.
Figure 6A. Measurement of the BT12, BT13, and ZH305 glioblastoma cell viability using the MTT-assay at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability. B. Measurement of the viability of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and embryonic kidney (HEK293T) cells at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability. C. Representative micrographs of BT12, BT13, and ZH305 glioblastoma cells treated with 1 µg/ml of clemastine for 24 h and stained with anti-galectin-1 (LGALS1) antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 20 µm. D. Representative micrographs of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and kidney cells (HEK293T) treated with 1 µg/ml clemastine for 24 h and stained with anti-LGALS1 antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.
a) The proportions of EGFP-positive SK-N-SH cells with EGFP-HDQ74 aggregates or cell death as in c, treated with or without 10 μM calpastatin, 50 μM N-acetyl-Leu-Leu-methional (ALLM) or 50 μM calpeptin for 48 h post-transfection.figf4(a) The proportions of EGFP-positive SK-N-SH cells with EGFP-HDQ74 aggregates or cell death as in Figure 1c, treated with or without 10 μM calpastatin, 50 μM N-acetyl-Leu-Leu-methional (ALLM) or 50 μM calpeptin for 48 h post-transfection. DMSO was control for ALLM and calpeptin. Error bars show 95% confidence interval. (b) HeLa cells were transfected with EGFP-HDQ74 construct along with siGLO (control siRNA) in presence or absence of calpain 1 or calpain 2 siRNA (1:3 ratio) for 96 h. EGFP- and siGLO-positive cells were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval. (c) HeLa cells, transfected with siGLO or siRNAs for calpain 1 or calpain 2 for 96 h, were analyzed with anti-calpain 1 or anti-calpain 2 domain III antibodies. (d) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with drugs (concentrations in a) for 96 h. Densitometry is relative to actin. Error bars show s.e.m. (e) A53T α-synuclein clearance in undifferentiated or nonmitotic neuronally differentiated (with 100 ng ml-1 nerve growth factor for 5 d) stable PC12 cells as in Figure 1a, treated with or without 10 μM calpastatin for 24 h. Densitometry is relative to actin. Error bars show s.e.m. (f) COS-7 cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active (CA) m-calpain (1:3 ratio) for 48 h, were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval. (g) SK-N-SH cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active m-calpain (1:3 ratio) for 4 h, were subsequently treated with DMSO (control), 1 μM verapamil or 1 μM clonidine for 48 h. The proportions of EGFP-positive cells with aggregates were assessed as in Figure 1c. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05.
Conditional knockout of Snai1 induces loss of CBC stem cellsSections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells (arrows depicting proliferating CBC stem cells at the base of crypts).Figure 3Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells (arrows depicting proliferating CBC stem cells at the base of crypts). Quantification of total PCNA‐positive cells per crypt and PCNA‐positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 (n = 4, P = 0.049, 0.026). Scale bars: 20 μm. The expression of 2 CBCstem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1KO small intestine. Lgr5 and Olfm4in situ hybridisation staining shows a loss of CBCstem cell marker expression after loss of Snai1 (high magnification inserts depict a representative crypt demarcated by a dotted line). Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry. Scale bars: 20 μm. The analysis of sorted and EGFP‐positive cells from Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1fl/fl and Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1+/+mice 5 days after βNF induction show an ˜tenfold reduction in Lgr5GFP‐positive CBCstem cells. Immunohistochemistry for active caspase‐3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt (n = 5, P = 0.0001) and villus (n = 5, P = 0.005) regions in Snai1KO tissue (arrows mark active caspase‐3‐positive cells). Scale bars: 20 μm. A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZCre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.
Assessment of TRAIL-R2 DISC stoichiometry.Figure 1(A) Western blot analysis of Caspase 8, FLIP and FADD recruitment to the TRAIL-R2 DISC in A549, HCT116 and DU145 cell lines following incubation with increasing concentrations (1x, 2x, or 4x) of anti-TRAIL-R2 (AMG655)-conjugated magnetic beads for 90 minutes. (B) Western blot analysis of the unbound soluble fraction An untreated input of each cell line is included for comparison. (C) Quantification of Western blot analysis for FLIP, FADD, Procaspase 8 and their respective cleavage fragments at the TRAIL-R2 DISC. Proteins were quantified by densitometry and normalised to known protein standards (D) Caspase-8-like (IETDase) activity assay in the unbound soluble fraction from the DISC IP An untreated control (0) was used for each cell line to show basal caspase-8 activity. (E) Ratios of DED proteins quantified at the TRAIL-R2 DISC in A549, HCT116 and DU145 cells; increasing concentrations of AMG655 are indicated by colour intensity. (F) Correlation between caspase-8 activity and ratio of FADD:FLIP(L).
Jumpy localizes to autophagic isolation membranes.Figure 4(A-H) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg16 (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and Atg16 (white in B and F or blue in D and H). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization among Jumpy CS, LC3 and Atg16, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg16, blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3. (I, J) C2C12 cells were transfected for 24 h with GFP (I), fixed and immunostained with anti‐Atg16 (J). (K-R) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg12(M) antibody. Jumpy CS (green), LC3 (red) and Atg12 (white in L, P or blue in N, R). Boxed areas (K-N) are shown at higher magnification in the corresponding panel below (O-R). White arrows indicate colocalization among Jumpy CS, LC3 and Atg12, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12, blue arrow indicates colocalization between Jumpy CS and Atg12 but not LC3. (S-ZA) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry‐LC3 (LC3), fixed and immunostained with anti‐lamp1. Jumpy CS (green), LC3 (red) and Lamp1 (white in T, X or blue in V, ZA). Boxed areas (S-V) are shown at higher magnification in the corresponding panel below (W-ZA). Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1. Scale bars, 2 μm.
SUMO modification of RPA appears independent of ssDNA length.Figure 10Histograms depicting SUMO conjugation rates to Rfa1 for different RPA-DNA complexes. Siz2 and DNA are both present in the reaction at a concentration of 1 µM while RPA concentration is varied from 1 to 3 µM and the length of single-stranded DNA is varied from 27 to 80 nucleotides. Representative gels are shown in Appendix Fig S7. A model for SUMO signal amplification. (i) Siz2 binds dsDNA and modifies with SUMO the RPA that is closest to the dsDNA/ssDNA junction. (ii) SUMO-modified RPA is exchanged with an unmodified RPA. (iii) Siz2 modifies the RPA that is closest to the dsDNA/ssDNA junction. (iv) SUMO-modified RPA is exchanged with an unmodified RPA.
Figure 3Heatmap showing unsupervised hierarchical clustering of human differentially expressed genes between human scr-shRNA and sh-TFEB ECs. Red: up-regulated genes; green: down-regulated genes. Selection of enriched functional GO categories by DAVID analysis in differentially expressed genes between human sh-TFEB and scr-shRNA ECs. GO analyses were performed individually on down‐ or up‐regulated genes using DAVID tool (biological process). GO terms are ranked by P‐value corrected by Benjamini Hockberg method, and the number of genes is indicated. Selection enriched of Molecular Pathways by GSEA analysis of microarray data comparing human sh-TFEB and scr-shRNA ECs. Normalized enrichment scores (NESs) and p-values are reported. Selection of enriched functional GO categories by DAVID analysis on ChiP-seq data set performed in human ECs overexpressing TFEBS142A. GO analyses were performed using DAVID tool (biological process). GO terms are ranked by P‐value corrected by BH method, and the number of genes is indicated.
a) EGF enhances colocalization of phosphorylated (P)-bRAF, P-MEK and P-ERK with LC3.f1a) EGF enhances colocalization of phosphorylated (P)-bRAF, P-MEK and P-ERK with LC3. Immunofluorescence (IF) showing colocalization of P-bRAF (green), P-MEK (green) and P-ERK (green) with LC3 (red) in NIH/3T3 cells in presence or absence of EGF (10 min as shown in scheme). The bars represent mean±s.e.m. **P0.01, ***P0.001; Student's t-test, 60 cells analysed from two experiments. Scale bars, 10 μm. (b) P-ERK colocalizes with pre-autophagosomal and autophagosomal structures. IF depicting colocalization of P-ERK (green or red) with LC3 (red), ATG5-ATG12 (red), ATG16 (green), vps34 (red), WIPI1 (red), WIPI2 (red) and P-ULK1 (red) in EGF-treated NIH/3T3 cells. Scale bars, 10 μm. The bars represent mean±s.e.m. 50 cells analysed from two experiments. (c) Growth factors in serum maintain P-ERK/LC3 colocalization. IF depicting colocalization of P-ERK (green) with LC3 (red) in 2 h serum-fed NIH/3T3 cells. (a-c) Native merged images or images with colocalization highlighted in white pixels are shown. Nuclei are blue (DAPI). Scale bar, 10 μm.
Figure 2Western blot showing STN1 knockdown. shLUC (siRNA targeting luciferase) was used as the control. CST deficiency causes ssDNA accumulation upon fork stalling. HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h, followed by 2 mM HU treatment for 3 h, and subsequently stained with BrdU antibody under a non-denaturing condition to detect ssDNA. N, the number of cells analyzed in each condition. Relative BrdU fluorescence intensity was quantitated by Image J. Scheme of DNA fiber analysis and representative fiber images. DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi-resistant Flag-STN1 WT re-expression with and without mirin treatment. DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment. DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment. DNA fiber analysis of U2OS cells showing that SMARCAL1-mediated fork reversal is needed for nascent-stand degradation observed in CST-deficient cells. DNA fiber analysis of U2OS cells showing that ZRANB3-mediated fork reversal is needed for nascent-strand degradation observed in CST-deficient cells.
Haploinsufficiency for E-cadherin in the presence of single allele β-catenin mutation leads to Wnt deregulation.Figure 5AhCreERCatnblox(ex3)/+ compared to AhCreER Catnblox(ex3)/+ Cdh1fl/+ at day 10 post-induction. Note the presence of colonic lesions in the colon of AhCreER Catnblox(ex3)/+ Cdh1fl/+ mice (arrows). Scale bar, 100 μm; red bar indicates proliferative zone (BrdU). Scoring of BrdU-positive cells per half-crypt in the small intestine of wild-type, AhCreERCdh1fl/+, AhCreERCatnblox(ex3)/+ and AhCreERCatnblox(ex3)/+Cdh1fl/+mice at day 10 post-induction. N ≥ 3, at least 25 crypts per mouse were scored. There was significantly higher proliferation in the AhCreERCatnblox(ex3)/+Cdh1fl/+mice (P = 0.028, one-sided Mann-Whitney U-test). In vitro growth of crypts (small intestine) from mice as indicated at day 5 post-induction without R-spo1. Quantification of sphere-forming efficiency of AhCreERCatnblox(ex3)/+AhCreERCatnblox(ex3)/+Cdh1fl/+, day 5 post-induction. N = 3 mice per genotype, mean of 6 technical replicates per mouse, P = 0.04 one-sided Mann-Whitney U-test. Survival of Lgr5CreERCatnblox(ex3)/+ and Lgr5CreERCatnblox(ex3)/+Cdh1fl/+ shows significant acceleration (P = 0.000123, log-rank test) after E-cadherin reduction. About 85% (6/7 mice) had colonic lesions, as identified with β-cateninIHC in contrast to Lgr5CreERCatnblox(ex3)/+mice.
Figure 7A. Representative electron micrographs showing a small/clear vesicle (SV, arrow), dense-core vesicles (DCVs, arrowheads) and a clathrin-coated pit (hatched arrow) in DRG neurons. B, C. Diameter distributions of SVs and DCVs (statistics from 6 control cells and 9 KD cells) in DRG neurons. The red and green lines are curves fitted to a Gaussian function. D, E. Diameters of SVs and DCVs in Syt11 KD and control neurons with (100K, statistics from 6 control cells and 9 KD cells) or without 100 mM K+ stimulation (SE, statistics from 6 cells for each group).
Fig.Figure 5A,B) ABF2-mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1, and varying exogenous ABA concentrations. Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. represent Replicates were normalized to controls without SnRK2 in (A, B) C) Maximum ABA response recovery of samples shown in (A,B) relative to the reporter activity of 0.1 mM ABA-treated yeast cells expressing SnRK2s but no ABI1. Data information: Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in (C). D-F) Analysis of the ABA response mediated by OST1 and SnRK2.4 using the receptors RCAR1 (D), RCAR8 (E), and RCAR14 (F). Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. The data in (D-F) are the mean ± s.e.m; n = 12, biological replicates from four independent yeast transformants for OST1 + RCAR1/RCAR8, and SnRK2.4 + RCAR8.
a) HeLa cells were transfected with 0.figf6(a) HeLa cells were transfected with 0.2 μg GFP-LC3 and 0, 0.2 or 0.6 μg of Bif-1SH3-Myc expression plasmids for 24 h. The total amount of plasmid DNA was adjusted at 0.8 μg per transfection with the pcDNA3 vector. The cells were then cultured in EBSS or complete medium for 3 h and the number of GFP-LC3 dots per GFP-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 40). (b) HeLa cells were transfected with GFP-LC3 in combination with either Bif-1SH3-HcRed, Endophilin A1SH3-HcRed or empty HcRed vector for 20 h. The cells were then cultured in 2 h or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was counted using a fluorescence microscope (mean ± s.d.; n = 46). Statistical significance in a and b was determined by Student's t-test and the asterisks indicate P 0.0001. (c) 293T cells were transfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc and anti-Flag polyclonal antibodies. Uncropped images of the immunoblots in c are shown in the Supplementary Information, Fig. S5.
Macro- and micro-ER-phagy are parallel pathways with distinct morphologies and molecular requirements.Figure 4A Electron micrograph of wild-type yeast treated with DTT for 3 h. Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies (white arrows). Cy, cytosol; V, vacuole. B Relative activity of cyto-Pho8∆60 after nitrogen starvation, which reports on non-selective macroautophagy of cytosol. Mean + SEM, n ≥ 3. WT, wild-type. Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after nitrogen starvation (C) Mean + SEM, n ≥ 3. Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after tunicamycin treatment (D, E). Mean + SEM, n ≥ 3. F, G Growth assays of untreated and tunicamycin-treated WT, ∆atg39/40, ∆snf7 and ∆snf7 ∆atg39/40 cells. Numbers indicate growth relative to WT cells based on the areas under the curves. Mean ± SEM, n = 3.
Figure 8Depolarisation of the mitochondrial membrane stabilises Pink1 in the outer membrane. However, without calcium, Miro remains protected and cannot be ubiquitinated by Parkin. Calcium binding to Miro1 EF hands removes the structural constraint preventing access of Parkin to its preferred lysine(s) or/and enables the phosphorylation of ubiquitin(s) on Miro. This amplifies the Parkin activation cycle and ensures faster Parkin translocation to mitochondria
Figure 6A, B NSG mice were pre-implanted with tumor fragments derived from two different PDX models: (A) TM00199 (L858R-EGFR; from The Jackson Laboratory) or (B) TM00193 (E746_A750 del-EGFR; from The Jackson Laboratory). Once tumors reached approximately 300 mm3, mice were treated with monoclonal antibodies (2XmAbs, cetuximab plus trastuzumab, 200μg/injection) twice a week, or with EGFR TKIs, erlotinib (50mg/kg) or osimertinib (10mg/kg) daily. Alternatively, mice were treated with a combination of 2XmAbs and either erlotinib or osimertinib, for either 32 days (A) or 42 days (B). Tumor growth was monitored twice a week. the antibodies were injected into the peritoneum and the TKIs were delivered orally. The figure shows tumor volumes of individual mice and the respective survival curves (right hand panels). The grey areas mark time windows of animal treatment. (C) Mice presented in the erlotinib+2XmAbs group were re-treated with the same drug combination (2XmAbs plus erlotinib) after a holiday period (>136 days), during which they received no treatment. Note that different colors identify individual mice
Confocal imaging and three-dimensional model reconstruction of neuron transfected with GFP-LC3.Figure 1  (A-J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A-D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A-D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K-O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M-O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N-O).
Figure 5(A-C) Single-particle-tracking of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Atto633-conjugated Fab fragments against CD22, CD19, IgM and IgD, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(A) Trajectories of CD22 showing diffusion of single particles over 6 s.(B) Magnified region from (A) traces one particle (current position indicated with white dashed circle, trajectory in red) over 6 s.(C) Diffusion coefficients of 300 representative CD22, CD19, IgM and IgD particles. Bars indicate the median. Data are pooled from at least three experiments per receptor. *P < 0.0001 (Student's t-test). (D-F) Single-particle-tracking of CD22 in primary LifeAct-GFP-expressing B cells. Cells were labelled with Atto633-conjugated Fab fragments against CD22, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(D) Trajectories of CD22 (left) showing diffusion of single particles over 6 s and overlaid with a still image of F-actin intensity (middle). Areas of high F-actin density (grey line boundaries) with trajectories of CD22 classified as being inside (red) and outside (blue) of F-actin rich areas.(E) Diffusion coefficients of CD22 particles from (D) whose trajectories were classed as inside (in) or outside (out) of actin rich areas. The overall diffusion is also shown (all). Bars and numbers are the median. Data are pooled from two different experiments. *P < 0.0001 (Student's t-test).(F) Correlation analysis. For each position in each CD22 track, the actin intensity was measured and compared to the same number of random positions on the same cell. Values from different cells were normalized so that the average intensity in F-actin rich regions corresponds to a value of one and the average for F-actin low regions to zero. (G) Wild-type primary B cells were labelled with Atto633-conjugated Fab fragments against CD22, settled onto nonstimulatory coverslips and treated with vehicle control (DMSO) or 1µM LatA. Single particle trajectories were then analysed. Bars and numbers indicate the median. Data are pooled from two experiments.*P < 0.0001 (Student's t-test). (H-J) Wild-type primary B cells were stained with Alexa647-conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(H) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(I-J) Quantification of the distribution of CD22 Hopkins index and with H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM.Data are pooled from two different experiments. (K) Flow cytometric analysis of F-actin by intracellular phalloidin staining of unstimulated or LatA treated wild-type B cells for the indicated length of time. (L) Flow cytometric analysis of CD22 surface expression in resting or stimulated B cells with 10µg/ml anti-µ chain (Jackson ImmunoResearch), 1µM or 10µM LatA for 5 and 15 minutes. Error bars denote mean ± SD.