Journal: NPJ Vaccines

Article Title: Epitope-specific immunity against Staphylococcus aureus coproporphyrinogen III oxidase

doi: 10.1038/s41541-020-00268-2

Figure Lengend Snippet: a S. aureus CgoX structure was modulated from B. subtillis (3I6D.pdb). The linear epitope of mAb D3 (red) was identified by microarray technology using overlapping 13mer CgoX peptides (Supplementary Fig. ). Protein structure was visualised by EzMol2.1. b Alanine scan of epitope peptides for binding analysis of anti-CgoX mAb D3. Single amino acid positions of the D3 epitope were consecutively replaced by alanine (red in right panel). Immobilised peptides were stained by anti-CgoX mAb D3 detected with anti-mIgG-HRP. Data are presented as mean ± s.d. ( n = 2 technical replicates). c Allele frequencies of anti-CgoX mAb D3 epitope. Genome sequences of S. aureus clinical isolates were analysed for epitope aa sequence using the RidomSeqsphere core genome multi locus sequence typing (cgMLST) database. Amino acids interacting with paratope of anti-CgoX mAb D3 according to alanine scan are marked in red. Amino acid differences from identified epitope peptide sequence are marked in blue. Frequencies of alleles with non-restricted binding of anti-CgoX mAb D3 are marked in green. d Uniqueness of the CgoX D3 epitope in S. aureus . Sequence alignment of CgoX from S. aureus with PPOX from H. sapiens and M. musculus . CgoX D3 epitope is depicted in yellow. e Competition analysis of CgoX mAb. Binding of DyLight-649-conjugated anti-CgoX mAb D3 to rCgoX was competed for with different concentrations of unconjugated, indicated mAbs and analysed by ELISA. Binding was determined by fluorescence measurement (Ex 646/Em 674). Data are presented as mean ± s.d. ( n = 2). f Saturation binding curve was generated by plotting absorbance signals (OD 450nm ) of increasing amounts of anti-CgoX huMAb D3 to rCgoX coated on ELISA MaxiSorp plate using the GraphPadPrism 8.4 software. Kd was calculated by non-linear fitting and the equation for one-site binding model [Y = Bmax*X/(Kd + X)].

Article Snippet: Upon washing with TBST (3 × 15 min), HRP conjugated secondary goat anti-mouse IgG (Sigma-Aldrich, Germany, cat. No. A3673) or goat anti-human IgG antibody (Bio-Rad, Germany, cat. No. 172-1050) were diluted 1:3,000 and applied for 1 h at room temperature, respectively.

Techniques: Microarray, Binding Assay, Staining, Sequencing, Enzyme-linked Immunosorbent Assay, Fluorescence, Generated, Software

Journal: NPJ Vaccines

Article Title: Epitope-specific immunity against Staphylococcus aureus coproporphyrinogen III oxidase

doi: 10.1038/s41541-020-00268-2

Figure Lengend Snippet: a Competition ELISA. Binding of anti-CgoX mAb D3 to rCgoX coated on ELISA MaxiSorp plate was competed for with CgoX-D3-BSA conjugate. Binding was detected with anti-mIgG-HRP and compared to control sample (BSA). b Anti-CgoX-BSA IgG titer. Sera of two CgoX-D3-BSA immunised mice were collected at day 68 and analysed together with preimmune serum for anti-CgoX IgGs by ELISA. c Survival of mice challenged with S. aureus upon immunisation with CgoX-D3 epitope peptide conjugated with BSA. BALB/c mice ( n = 11) immunised with CgoX-D3-BSA or the carrier protein BSA (black) as control group ( n = 10), were infected i.p. with 3.3 × 10 7 cfu S. aureus USA300 mixed with 5% mucin from porcine stomach. Significance was calculated by Log-rank (Mantel-Cox) test.

Article Snippet: Upon washing with TBST (3 × 15 min), HRP conjugated secondary goat anti-mouse IgG (Sigma-Aldrich, Germany, cat. No. A3673) or goat anti-human IgG antibody (Bio-Rad, Germany, cat. No. 172-1050) were diluted 1:3,000 and applied for 1 h at room temperature, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Infection

Journal: bioRxiv

Article Title: An integrated signaling threshold initiates IgG response towards virus-like immunogens

doi: 10.1101/2024.01.28.577643

Figure Lengend Snippet: ( a ) Representative single-molecule fluorescence trajectories from Alexa-594 labeled RBD-specific Fab (Alexa-594-Fab). We prepared Alexa-594-Fab using a monoclonal IgG2a specific for SARS-CoV-2 RBD (BioLegend #944803, mAb1 herein). This high-affinity Fab allowed us to quantitate epitope density (ED) on individual SVLS conjugated with RBD, using single-molecule fluorescence techniques that we established previously – . Briefly, the Fab was prepared from the IgG form of the antibody using a Pierce TM Fab Micro preparation kit (ThermoFisher Scientific, CAT#44685) following manufacturer instructions. The purified Fab was then labeled using Alexa Fluor 594 succinimidyl NHS ester (ThermoFisher Scientific, CAT#A37572) following the protocol that we established previously . The labelling ratio between the dye and protein was determined to be 3.3 dye molecules per Fab based on single-molecule two-photon fluorescence measurement as we described previously . As shown in in the main text, we deposited pRBD(DNA1)4 bound with Alexa-594-Fab on poly-L-lysine coated coverslips and used epi-fluorescence imaging to directly visualize individual SVLS through excitation of the Alexa-594 fluorophore by a 592-nm laser. Individual SVLS showed up as bright spots in a dark background. Neither SVLS alone nor free Alexa-594-Fab alone at the same concentrations yielded these bright spots under the microscope. Moreover, no fluorescent spots were visible when Alexa-594-Fab was incubated with control SVLS without RBD conjugation, confirming that these fluorescent spots were specific for SVLS conjugated with RBD but not protein aggregates. In order to determine the number of RBD molecules per SVLS, we chose to work at a laser power of 100 mW. This illumination condition allowed us to collect the initial fluorescence intensity from individual spots, and the constant illumination of the sample under this condition also allowed us to observe the photobleaching of individual fluorophores with time. As shown in , these photobleaching events display a hallmark feature of ‘steps’, where the fluorescence persists for a finite time followed by a sudden decrease in fluorescence intensity. These steps correspond to the photobleaching of individual molecules. We have used a step-detection algorithm that we developed previously to identify steps from these real-time fluorescence traces. Measurement of this ‘step’ size yields the fluorescence intensity of a single Alexa-594 fluorophore, which can be used as an internal reference to convert the initial fluorescence intensity of individual liposomes to the number of Alexa-594 fluorophores, as we have done previously for individual HIV-1 virions and individual protein-conjugated liposomes using the same quantitation methodology. ( b ) The histogram of the individual photobleaching step sizes for Alexa-594 molecules (N=247), which could be well described by the sum of two Gaussians (red curve), one centered at 630 ± 15 analog-to-digital units (a.u., mean ± standard error) and the other centered at 1249 ± 40 a.u. Under a statistical significance level of 5%, Pearson’s Chi-square test selected the double-Gaussian distribution (P-value=0.88) and rejected the single-Gaussian distribution (P-value=0.02) as the model to describe the data. The difference in peak values is close to twofold. As we have observed previously, the secondary peak may result from the photobleaching of two Alexa-594 molecules that occurred almost simultaneously, which could not be resolved by either the finite camera exposure time or the step-finding algorithm , . The fluorescence intensity of a single Alexa-594 fluorophore can then be used to calculate the total number of Alexa-594 molecules per SVLS based on a ratio comparison , with the initial fluorescence intensity of Alexa-594 associated with individual SVLS. The number of RBD molecules per SVLS was then calculated as the total number of Alexa-594 molecules per SVLS normalized by the average number of Alexa-594 molecules per Fab. ( c ) The distributions of ED for pRBD(DNA1)1 (orange), pRBD(DNA1)2 (red), pRBD(DNA1)3 (green), and pRBD(DNA1)4 (blue). These distributions can be well described with Gaussian probability density function, with means and standard deviations of 5 ± 2 for pRBD(DNA1)1 (N=204), 15 ± 4 for pRBD(DNA1)2 (N=182), 31 ± 9 for pRBD(DNA1)3 (N=185), and 43 ± 10 for pRBD(DNA1)4 (N=217), respectively. These values are very close to the value of ED estimated using the ensemble approach as we established previously – , .

Article Snippet: To determine the subclasses of IgG antibodies elicited in mice upon immunization by various agents, we used the following secondary antibodies during ELISA: goat anti-mouse IgG1 HRP antibody (#1071-05, SouthernBiotech), goat anti-mouse IgG2a HRP antibody (#1081-05, SouthernBiotech), goat anti-mouse IgG2b HRP antibody (#1091-05, SouthernBiotech), goat anti-mouse IgG2c HRP antibody (#1078-05, SouthernBiotech), and goat anti-mouse IgG3 HRP antibody (#1101-05, SouthernBiotech).

Techniques: Fluorescence, Labeling, Purification, Imaging, Microscopy, Incubation, Control, Conjugation Assay, Liposomes, Quantitation Assay, Comparison

Journal: bioRxiv

Article Title: An integrated signaling threshold initiates IgG response towards virus-like immunogens

doi: 10.1101/2024.01.28.577643

Figure Lengend Snippet: ( a )( b ) RBD-specific IgG (a) or HEL-specific IgG (b) in mouse sera of B6 genetic background measured by ELISA after a single injection using various agents listed in the insets. ( c ) Neutralization of HIV-1 virions pseudotyped with SARS-CoV-2 S protein by mouse sera from various conditions in (a). ( d ) through ( i ): ELISA detection for RBD- and HEL-specific IgG subclasses in sera samples from (a) and (b). The y-axes for panels (d), (e), (f), (h) and (i) are shown in OD450 for 100-fold diluted sera because reference Ag-specific IgG subclasses are not available. ( j ) RBD-specific IgG (left) or HEL-specific IgG (right) in BALB/c mouse sera measured by ELISA after a single injection using various agents #1 through #7 as listed in (a) and (b) insets, respectively. Throughout , data from D5 post immunization are shown in circles and data from D11 are shown in columns; the doses of RBD and HEL were 0.24 and 0.3 µg per animal, respectively for Conditions 2 through 10. The pairwise statistical difference between each condition and Condition 1 on Day 5 was determined by Student’s T-test and shown using the following set of symbols; **: p-value < 0.01; *: p-value < 0.05; NS: not significant, p-value > 0.05. N=4.

Article Snippet: To determine the subclasses of IgG antibodies elicited in mice upon immunization by various agents, we used the following secondary antibodies during ELISA: goat anti-mouse IgG1 HRP antibody (#1071-05, SouthernBiotech), goat anti-mouse IgG2a HRP antibody (#1081-05, SouthernBiotech), goat anti-mouse IgG2b HRP antibody (#1091-05, SouthernBiotech), goat anti-mouse IgG2c HRP antibody (#1078-05, SouthernBiotech), and goat anti-mouse IgG3 HRP antibody (#1101-05, SouthernBiotech).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Neutralization

Journal: bioRxiv

Article Title: An integrated signaling threshold initiates IgG response towards virus-like immunogens

doi: 10.1101/2024.01.28.577643

Figure Lengend Snippet: (a) and (b) We used mAb1, the commercial RBD-specific monoclonal mouse IgG2a (BioLegend CAT#944803) as the known reference to construct standard curves for ELISA to indicate the sensitivity of current ELISA assays for detection of RBD-specific IgG and IgG2a. (c) and (d) We used mAb2, the HEL-specific monoclonal mouse IgG1 (clone HyHEL10, a special gift from Prof. Irina Grigorova) as the known reference to construct standard curves for ELISA to indicate the sensitivity of current ELISA assays for detection of HEL-specific IgG and IgG1. The results are representative of three or more independent repeats of the same experiments. The data were all fit to Richard’s five-parameter dose-response curves (black curves throughout ) in GraphPad Prism 9 for interpolation of Ag-specific IgG concentrations .

Article Snippet: To determine the subclasses of IgG antibodies elicited in mice upon immunization by various agents, we used the following secondary antibodies during ELISA: goat anti-mouse IgG1 HRP antibody (#1071-05, SouthernBiotech), goat anti-mouse IgG2a HRP antibody (#1081-05, SouthernBiotech), goat anti-mouse IgG2b HRP antibody (#1091-05, SouthernBiotech), goat anti-mouse IgG2c HRP antibody (#1078-05, SouthernBiotech), and goat anti-mouse IgG3 HRP antibody (#1101-05, SouthernBiotech).

Techniques: Construct, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: An integrated signaling threshold initiates IgG response towards virus-like immunogens

doi: 10.1101/2024.01.28.577643

Figure Lengend Snippet: (a) through (d) ELISA measurements for RBD-specific IgG1 (a), IgG2a (b), IgG2b (c), and IgG3 (d) in mouse sera after a single subcutaneous injection using various agents listed in (a) inset with no additional adjuvant. The dose of RBD per injection was 0.24 µg for conditions 2 through 7. (e) through (h) ELISA measurements for HEL-specific IgG1 (e), IgG2a (f), IgG2b (g) and IgG3 (h) in mouse sera after a single subcutaneous injection using various agents listed in (e) inset with no additional adjuvant. The dose of HEL per injection was 0.3 µg for conditions 2 through 7. Throughout , data from D5 post immunization are shown in circles and data from D11 are shown in columns. The pairwise statistical difference between each condition and Condition 1 on Day 5 was determined by Student’s T-test and shown using the same set of symbols as . N=4. The y-axes for all panels except (b) and (e) are shown in OD450 for 100-fold diluted sera because reference Ag-specific IgG subclasses are not available.

Article Snippet: To determine the subclasses of IgG antibodies elicited in mice upon immunization by various agents, we used the following secondary antibodies during ELISA: goat anti-mouse IgG1 HRP antibody (#1071-05, SouthernBiotech), goat anti-mouse IgG2a HRP antibody (#1081-05, SouthernBiotech), goat anti-mouse IgG2b HRP antibody (#1091-05, SouthernBiotech), goat anti-mouse IgG2c HRP antibody (#1078-05, SouthernBiotech), and goat anti-mouse IgG3 HRP antibody (#1101-05, SouthernBiotech).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Adjuvant

Journal: bioRxiv

Article Title: An integrated signaling threshold initiates IgG response towards virus-like immunogens

doi: 10.1101/2024.01.28.577643

Figure Lengend Snippet: ( a ) and ( c ) ELISA measurements for RBD-specific IgG (a) or HEL-specific IgG (c) in B6 genetic background after a single injection using various agents listed in respective insets. ( b ) and ( d ) Titers for RBD-specific IgG (b) or HEL-specific IgG (d) measured for various conditions as in (a) and (c). ( e ) Neutralization of HIV-1 virions pseudotyped with SARS-CoV-2 S protein by mouse sera from various conditions in (a). ( f ) Titer values for RBD-specific IgG (left) or HEL-specific IgG (right) in BALB/c mice after a single injection using agents #4, 5, 6, 7 and 15 as listed in (a) and (c) insets. Throughout , data from D5 post immunization are shown in circles and data from D11 are shown in columns or squares; the doses of RBD and HEL were 0.24 and 0.1 µg per animal, respectively for Conditions 1 through 15; the pairwise statistical difference between each condition and Condition 1 on Day 5 was determined by Student’s T-test and shown using the following set of symbols; ***: p-value < 0.001; **: p-value < 0.01; *: p-value < 0.05; NS: not significant, p-value > 0.05. N=4.

Article Snippet: To determine the subclasses of IgG antibodies elicited in mice upon immunization by various agents, we used the following secondary antibodies during ELISA: goat anti-mouse IgG1 HRP antibody (#1071-05, SouthernBiotech), goat anti-mouse IgG2a HRP antibody (#1081-05, SouthernBiotech), goat anti-mouse IgG2b HRP antibody (#1091-05, SouthernBiotech), goat anti-mouse IgG2c HRP antibody (#1078-05, SouthernBiotech), and goat anti-mouse IgG3 HRP antibody (#1101-05, SouthernBiotech).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Neutralization

Journal: bioRxiv

Article Title: An integrated signaling threshold initiates IgG response towards virus-like immunogens

doi: 10.1101/2024.01.28.577643

Figure Lengend Snippet: ELISA detection for RBD-specific (a through d) and HEL-specific (e through h) IgG subclasses for sera from conditions listed in and , respectively. The y-axes for all panels except (e) are shown in OD450 for 100-fold diluted sera because reference Ag-specific IgG subclasses are not available. Throughout , data from D5 post immunization are shown in circles and data from D11 are shown in columns. The pairwise statistical difference between each condition and Condition 1 on Day 5 was determined by Student’s T-test and shown using the following set of symbols; ***: p-value < 0.001; **: p-value < 0.01; *: p-value < 0.05; NS: not significant, p-value > 0.05. N=4.

Article Snippet: To determine the subclasses of IgG antibodies elicited in mice upon immunization by various agents, we used the following secondary antibodies during ELISA: goat anti-mouse IgG1 HRP antibody (#1071-05, SouthernBiotech), goat anti-mouse IgG2a HRP antibody (#1081-05, SouthernBiotech), goat anti-mouse IgG2b HRP antibody (#1091-05, SouthernBiotech), goat anti-mouse IgG2c HRP antibody (#1078-05, SouthernBiotech), and goat anti-mouse IgG3 HRP antibody (#1101-05, SouthernBiotech).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: An integrated signaling threshold initiates IgG response towards virus-like immunogens

doi: 10.1101/2024.01.28.577643

Figure Lengend Snippet: ( a ) through ( d ) ELISA measurements for RBD-specific IgG1 (a), IgG2a (b), IgG2b (c) and IgG3 (d) antibody in mouse sera after a single subcutaneous injection using various agents listed in (a) inset with no additional adjuvant. The dose of RBD per injection was 0.24 µg for conditions 1 through 6. ( e ) through ( h ) ELISA measurements for HEL-specific IgG1 (e), IgG2a (f), IgG2b (g) and IgG3 (h) antibody in mouse sera after a single subcutaneous injection using various agents listed in (e) inset with no additional adjuvant. The dose of HEL per injection was 0.1 µg for conditions 1 through 6. The y-axes for all panels except (b) and (e) are shown in OD450 for 100-fold diluted sera because reference Ag-specific IgG subclasses are not available. Throughout , data from D5 post immunization are shown in circles and data from D11 are shown in columns. The pairwise statistical difference between each condition and Condition 1 on Day 5 was determined by Student’s T-test and shown using the same set of symbols as . Error bars represent the standard errors (N=4). The dose of DNA1 in the admixture with soluble proteins was 1 nmol.

Article Snippet: To determine the subclasses of IgG antibodies elicited in mice upon immunization by various agents, we used the following secondary antibodies during ELISA: goat anti-mouse IgG1 HRP antibody (#1071-05, SouthernBiotech), goat anti-mouse IgG2a HRP antibody (#1081-05, SouthernBiotech), goat anti-mouse IgG2b HRP antibody (#1091-05, SouthernBiotech), goat anti-mouse IgG2c HRP antibody (#1078-05, SouthernBiotech), and goat anti-mouse IgG3 HRP antibody (#1101-05, SouthernBiotech).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Adjuvant

Journal: bioRxiv

Article Title: An integrated signaling threshold initiates IgG response towards virus-like immunogens

doi: 10.1101/2024.01.28.577643

Figure Lengend Snippet: BCR-mediated endocytosis can occur to all particles depicted above, from low to high ED. However, for SVLS without iNA, both CD19 (slim orange rectangles) and a threshold of ED are required to meet the signaling threshold required for CSR and IgG secretion. For SVLS with iNA, CD19 can be bypassed for CSR and IgG secretion because the combined signal from BCR and intrinsic TLR activation can be sufficient to overcome the signaling threshold. Note that BCR and intrinsic TLR activation converge at or before CSR and IgG secretion take place. In response to virus-like immunogens, CSR and IgG secretion can occur independently of cognate T-cell help or TLR activation. Also, Ag-specific B cells may undergo proliferation and/or differentiation before CSR and IgG secretion, which is noted above.

Article Snippet: To determine the subclasses of IgG antibodies elicited in mice upon immunization by various agents, we used the following secondary antibodies during ELISA: goat anti-mouse IgG1 HRP antibody (#1071-05, SouthernBiotech), goat anti-mouse IgG2a HRP antibody (#1081-05, SouthernBiotech), goat anti-mouse IgG2b HRP antibody (#1091-05, SouthernBiotech), goat anti-mouse IgG2c HRP antibody (#1078-05, SouthernBiotech), and goat anti-mouse IgG3 HRP antibody (#1101-05, SouthernBiotech).

Techniques: Activation Assay, Virus

Journal: Cell reports

Article Title: Post-Golgi carriers, not lysosomes, confer lysosomal properties to pre-degradative organelles in normal and dystrophic axons

doi: 10.1016/j.celrep.2021.109034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Subsequent staining steps were performed according to manufacturer’s instructions (Vector Laboratories), which include incubation with biotinylated secondary antibodies, complex formation with avidin and biotinylated HRP using Vectorstain ABC Kit, and reaction with Vector DAB Peroxidase Substrate.

Techniques: Plasmid Preparation, Electron Microscopy, Recombinant, Immunodetection, Fluorescence, Transfection, Expressing, Software

Journal: Lab on a Chip

Article Title: Label-free ferrohydrodynamic separation of exosome-like nanoparticles

doi: 10.1039/d0lc00609b

Figure Lengend Snippet: Figure 5. Characterization of the FerroChip in separating exosome-like nanoparticles from biological samples. a We chose to use a 1 µL min-1 sample flow rate in order to separate exosome-like nanoparticles (30 – 150 nm in diameter) from extracellular media and human serum. This sample flow rate maximized the separation between exosome-like nanoparticles and large EVs (200 – 1000 nm in diameter). The ratio of sample flow and sheath flow was 1:5 (sample – 1 µL min-1; sheath –5 µL min-1). Other device and operating parameters remained the same as in previous figures. b Super-resolution microscopic images of exosome-like nanoparticles (PKH 67 green fluorescence) and large EVs (PKH 26 red fluorescence) from the mixture (pre-separation), and the samples after FerroChip processing (large EVs and exosome outlets). A total of 730 particles were analyzed. c Western blot analysis of CD63 and HSP70 protein levels in large EVs and exosome-like nanoparticles that were processed by the FerroChip. d Transmission electron microscopy (TEM) images of exosome-like nanoparticles collected from the FerroChip. e Size distribution of separated exosome-like nanoparticles from TEM images (n=755). f Immunofluorescence images of separated exosome-like nanoparticles from human serum. Three channels were used in immunofluorescent staining, including EpCAM (green), CD24 (red), and CD63 (cyan). g Venn diagram depicts the percentage of antibody presence on the surfaces of separated exosome-like nanoparticles (n=12000) from human serum. EpCAM+ alone: 5%; CD24+ alone: 52%; CD63+ alone: 21%; EpCAM+ and CD24+:4%; EpCAM+ / CD63+: 2%; CD24+ / CD63+: 14%; CD63+ / CD24+ / EpCAM+: 2%.

Article Snippet: The membrane was then incubated overnight with exosome primary antibodies against CD63 and HSP70 (Santa Cruz Biotechnology, Inc., Dallas, TX following incubation with Goat-Rabbit-HRP secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX).

Techniques: Fluorescence, Western Blot, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: A Ragulator–BORC interaction controls lysosome positioning in response to amino acid availability

doi: 10.1083/jcb.201703094

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: GFP-HRP , Miltenyi Biotec , 130-091-833.

Techniques: FLAG-tag

Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. SSTR3 fused to the fluorescent protein mNeonGreen (NG) at its intracellular C-terminus and a biotinylation Acceptor Peptide (AP) tag at its extracellular N-terminus was expressed under the control of an attenuated EF1α promoter (pEF1α Δ ) in wild type (WT) and Arl6 -/- IMCD3 cells. Cells were treated with or without somatostatin-14 (sst) for 2 h, then fixed and stained for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). AP SSTR3 NG (cyan) was imaged through the intrinsic fluorescence of NG. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar: 5μm (main panel), 2μm (inset). In WT cells, the ciliary SSTR3 signal decreases over the experimental time course. In Arl6 -/- cells, SSTR3 fails to exit cilia and an increase in the ciliary Ub level is detected. As a control, Arl6 -/- cells that did not express SSTR3-NG were tested; no increase in ciliary Ub levels was observed upon addition of sst. B. The fluorescence intensity of the Ub channel in the cilium was measured in each condition and the data are represented as violin plots. The thick bar indicates the median and the dotted lines the first and third quartiles. An 11-fold increase in ciliary Ub signal is observed upon addition of sst to SSTR3-expressing cells. Asterisks indicate ANOVA significance value. ****, p= <0.0001. C. WT or Arl6 -/- IMCD3 cells stably expressing AP SSTR3 NG and the biotin ligase BirA targeted to the ER lumen (BirA-ER) were transiently transfected with HA-tagged ubiquitin (HA-Ub). 10 µM biotin was added to cells 24 h post transfection for maximal biotinylation of AP SSTR3 NG and, after another 18 h, cells were treated with sst (10μM) for indicated times. Cells were lysed under denaturing conditions and biotinylated SSTR3 was captured on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6 and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. D. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in C was repeated three times and for each experiment, Ub-SSTR3 signals were normalized to the value in Arl6 -/- cells at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values. E. IMCD3 cells of the indicated genotypes were treated with the Smoothened agonist SAG or the vehicle DMSO for 2h. Cells were then fixed and stained for acTub and Ub. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar 5μm (main panel), 2μm (inset). Activation of Hh signaling promotes a detectable increase in ciliary Ub levels only in Arl6 -/- cells. F. Violin plots of the fluorescence intensity of the Ub channel in the cilium in each condition are shown. Asterisks indicate ANOVA significance value. ****, p= <0.0001.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Control, Staining, Ubiquitin Proteomics, Fluorescence, Expressing, Stable Transfection, Transfection, Western Blot, Quantitation Assay, Activation Assay

Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. IMCD3 cells of the indicated genotypes expressing AP SSTR3 NG were treated with somatostatin-14 for 2 h. Cells were fixed and stained for AcTub (magenta) and with antibodies specific for the lysine 63 (UbK63) or lysine 48 (UbK48) Ub chain linkages (yellow). SSTR3 NG (cyan) was imaged through the intrinsic fluorescence of NG. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar 5μm (main panel), 2μm (inset). B. The fluorescence intensity of the UbK48 and UbK63 channels in the cilium are represented as violin plots. A 14-fold increase in ciliary Ub abundance is detected with the K63Ub linkage-specific antibody. Asterisks indicate ANOVA significance value. ****, p= <0.0001. No ciliary signal is detected with the K48Ub linkage-specific antibody. C. Arl6 -/- IMCD3-[ AP SSTR3; BirA-ER] cells were transfected with the HA-tagged ubiquitin variants WT, noK0 (all seven acceptor lysine residues mutated to arginine) or K63 (where all lysine residues are mutated to arginine except for K63). Biotin was included in the culture medium and cells were treated with sst for 0 or 10 min. Cells were lysed under denaturing conditions and biotinylated SSTR3 was captured on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6 and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. WT IMCD3 cells were processed in parallel as a control. D. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in C was repeated four times and for each Ub variant, Ub-SSTR3 signals were normalized to the value at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values. Asterisks indicate ANOVA significance value. *** p = < 0.001; ** p=<0.01.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Expressing, Staining, Fluorescence, Transfection, Ubiquitin Proteomics, Western Blot, Control, Quantitation Assay, Variant Assay

Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. IMCD3-[pEF1α Δ - AP SSTR3 NG ] cells of the indicated genotypes were treated with sst for 2h before fixation and staining for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). SSTR3 NG was visualized through the intrinsic fluorescence of NG (cyan). Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. SSTR3 exit is blocked in Arl6 -/- cells regardless of the β-Arrestin2 genotype but the ciliary Ub signal is only evident when β-Arrestin2 function is intact. Scale bar: 5μm (main panel), 1μm (inset). B. Violin plots representing the ciliary levels of Ub under the indicated conditions. Asterisks indicate ANOVA significance value. ****, p= <0.0001. C. IMCD3 WT, knockout for Arl6 only and double knockout for Arl6 and the β-Arrestin2 gene Arrb2 were treated with SAG for 2h before fixation and staining for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar: 5μm (main panel), 1μm (inset). D. Violin plots representing the ciliary Ub levels. Asterisks indicate ANOVA significance value. ****, p= <0.0001; **, p= <0.01. The appearance of a Ub signal in cilia in Arl6 -/- cells depends on β-Arrestin2. E. IMCD3-[ AP SSTR3; BirA-ER] cells of the indicated genotypes stably expressing AP SSTR3 NG and BirA-ER were transfected with HA-Ub, biotin was added to the medium and cells were treated with sst for indicated times. Biotinylated SSTR3 was captured from cell lysates under denaturing conditions on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6, β-Arrestin2 to verify genotypes and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. WT IMCD3 cells were processed in parallel as a control. F. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in E was repeated twice and for each experiment, Ub-SSTR3 signals were normalized to the value in Arl6 -/- cells at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Staining, Ubiquitin Proteomics, Fluorescence, Knock-Out, Double Knockout, Stable Transfection, Expressing, Transfection, Western Blot, Control, Quantitation Assay

Journal: Nutrition & Metabolism

Article Title: High-fructose diet induces depressive-like behaviors and short-term memory deficits through hippocampal neurogenesis impairment via neural stem cell dysfunction

doi: 10.1186/s12986-025-01043-7

Figure Lengend Snippet: High fructose promotes NSC ferroptosis. ( A ) GO enrichment analysis of DEGs between CTL and Fru96 group in NE-4c cells. ( B ) Heatmap of ferroptosis-related DEGs between CTL and Fru96 group in NE-4c cells. ( C ) Representative images of mito-Fe 2+ and mito-ROS levels in high-fructose-administered NE-4c cells. ( D ) Statistical results of mito-Fe 2+ and mito-ROS concentration in NE-4c mitochondria. ( E ) Representative images of NE-4c intracellular lipid peroxidation levels. BODIPY OX is used to label oxidized lipids within cells, and BODIPY RED is used to label total lipids within cells. ( F ) Fluorescence intensity of lipid peroxidation in NE-4c cells. Data are expressed as Mean ± Sem, *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: A lipid peroxidation kit (S0131, Beyotime) was used to test MDA content in NE-4c cells.

Techniques: Concentration Assay, Fluorescence