Journal: bioRxiv

Article Title: p125A (Sec23ip) couples COPII coat assembly with donor-acceptor membrane organization to facilitate tunnel-based traffic

doi: 10.1101/2025.05.07.652703

Figure Lengend Snippet: A-B. Permeabilized HeLa cells were either pre-incubated in a reaction buffer lacking nucleotides, cytosol or SAR1 proteins for 15 min at 32°C (rundown) or not (control). Subsequently, a full transport cocktail containing rat liver cytosol (RLC) and SAR1-GTP (hamster SAR1a-H79G, 5μg/220μL) were added as indicated. The reaction was carried out for 15 minutes at 32 ° C, the cells were washed, fixed and analyzed for Sec31(using a polyclonal anti Sec31A antibody, A .) or Sec24C ( B. ) at ERES as we described C. The intensity of Sec31A or Sec24C was quantified from 9 fields of cells (20-60 cells per field, each field represented by a circle) derived from 3 to 4 independent experiments. Controls were defined as 100 percent and results analyzed using two-tailed t-test. D. Control and rundown reactions were as described in A in the presence of SAR1-GTP and RLC. Fixed cells were processed for IF using anti Sec24C and Sec31A (monoclonal) antibodies as indicated. E. Permeabilized cells expressing Tac-gp25l (ER marker) were incubated under control or rundown conditions (as in A) with SAR1-GTP and RLC, fixed and stained for Sec13 (red) and Tac (green). F. Analysis of Sec31A recruitment to ERES (marked with anti Sec16A antibody) in permeabilized control or Sec16A-KD cells (staining control) supplemented with Sar1-GTP and RLC as in A was analyzed from 6 fields of cells per condition (circles) from two independent experiments using Pearson Correlation Coefficient (PCC) (shown with two tailed t-test) G. Cells expressing GFP-tagged Sec16A were permeabilized and incubated as in A with RLC and Sar1-GTP, fixed and stained for Sec24C (red). Arrows in inserts indicate juxtapose labeling of recruited Sec24C with ERES markers in control and rundown conditions . H. Reconstitution of COPII recruitment was conducted as described in A. The cells were fixed and labeled with antibodies to Sec31 (polyclonal) and Sec24C, further fixed and processed for EM ( , ). Bars are 5μm in A and 200nm in E. Arrows in E point to gold labeling of Sec24C or Sec31 at ERES as indicated.

Article Snippet: Anti FLAG antibodies (M2, F1804 Sigma), antibodies to Sec16A, Novus Biologicals (NB100-1799), antibodies to p97/VCP, RDI research diagnostics (RDI PR065278), antibodies to GFP, Polysciences Inc. (24240) and Proteintech (66002-1-ig).

Techniques: Incubation, Control, Derivative Assay, Two Tailed Test, Bioprocessing, Expressing, Marker, Staining, Labeling

Journal: bioRxiv

Article Title: p125A (Sec23ip) couples COPII coat assembly with donor-acceptor membrane organization to facilitate tunnel-based traffic

doi: 10.1101/2025.05.07.652703

Figure Lengend Snippet: Active Rab1 or Sec16A depletion do not rescue the linkage between COPII inner and outer layers. A. Cells were transfected with construct expressing Flag-Rab1a-GTP(Q70L), permeabilized and subjected to rundown and COPII recruitment as in , monitoring Sec13 (red) and Rab1-GTP (anti Flag, green). Note that Rab1a-GTP is retained in cells and occasionally preserves Sec13 at the sites. B. Sec16A was depleted using siRNA and analyzed by western blot with Sec31A serving as a control as indicated (triplicates of control or KD samples are shown in the left panel). Depleted cells were permeabilized and subjected to rundown pre-incubation. Subsequently, control or Sec16A depleted cells were supplemented with RLC and SAR1-GTP (as in ). The recruitment of the coat outer layer (Sec31A, monoclonal antibody) and Sec16A localization was determined by IF. Bars are 5 μm.

Article Snippet: Anti FLAG antibodies (M2, F1804 Sigma), antibodies to Sec16A, Novus Biologicals (NB100-1799), antibodies to p97/VCP, RDI research diagnostics (RDI PR065278), antibodies to GFP, Polysciences Inc. (24240) and Proteintech (66002-1-ig).

Techniques: Transfection, Construct, Expressing, Western Blot, Control, Incubation

Journal: Nature chemical biology

Article Title: Functional Dissection of the Retrograde Shiga Toxin Trafficking Inhibitor Retro-2

doi: 10.1038/s41589-020-0474-4

Figure Lengend Snippet: ( a ) Scheme of biorthogonal click chemistry adapted to Retro-2.1. The clickable Retro-2.1 (compound 2) , based on Retro-2.1 (compound 1 , Ref. , ) is coupled via the DIBO moiety to biotin (compound 3 ), or a fluorophore (compound 4 ). Blue/dashed boxes indicate reactive centers. ( b ) Anti-Sec16A western blot of a representative pull-down with the clickable Retro-2.1-probe (compound 5 ). Load corresponds to the western blot of the input fraction. See for full gels. ( c ) Quantification of four (control and Retro-2.1) and two (competition, Comp.) independent experiments of clickable Retro-2.1 pull-down shown in (b). Bead control and competition conditions normalized to Retro-2.1 pull-down. Graph shows mean +/- SD, each point represents an individual experiment. Unpaired two-tailed t-test comparing Retro-2.1 to control, *** P < 0.0001. ( d ) Characterization of the Sec16A 1266-1678 /Sec13-Retro-2.1 interaction using bio-layer interferometry. Association and dissociation steps after subtraction of control curves (loading step without biotinylated Retro-2.1 probe) and analysis of binding kinetics of Sec16A 1266-1678 /Sec13 to biotinylated Retro-2.1 (compound 5 ). Graph is representative of 2 independent experiments. Data were acquired repeatedly from the same sample. ( e ) Confocal images of clickable Retro-2.1 reacted with an Alexa488 probe (compound 6 ) on mock siRNA transfected (control) or Sec16A-depleted (siSec16A) cells. DNA was stained with DAPI. Scale bars = 10 μm. Image representative of 2 independent experiments. ( f ) Quantification of fluorescence intensity of clicked Retro-2.1 (compound 6) in the indicated conditions. Quantification of 97 (negative control), 114 (Retro-2.1) and 77 (siSec16A) cells per condition from 2 independent experiments. Each point represents one cell. Negative control is fluorophore in the absence of clickable Retro-2.1 (“No Retro-2.1”). Graph shows mean +/- SD. Unpaired two-tailed t-test performed to compare Retro-2.1 treated and Retro-2.1+siSec16A conditions, *** P < 0.0001. See for numbered compound synthetic procedures.

Article Snippet: Antibodies against the following antigens were purchased from the indicated sources: Syn5 (Synaptic Systems, Cat#110053), GFP, giantin (institutional antibody platform at Curie Institute), TGN46 (BioRad, Cat#AHP500GT) GS27, GS28 (both BD Biosciences; Cat#611034, and Cat#611184), Sec16A (Proteintech Europe, Cat#20025-1-AP).

Techniques: Western Blot, Control, Two Tailed Test, Binding Assay, Transfection, Staining, Fluorescence, Negative Control

Journal: Nature chemical biology

Article Title: Functional Dissection of the Retrograde Shiga Toxin Trafficking Inhibitor Retro-2

doi: 10.1038/s41589-020-0474-4

Figure Lengend Snippet: ( a ) HeLa cells transfected with the indicated siRNAs, or treated for 30 min with 25 μM Retro-2, followed by incubation for 45 min at 37 °C with STxB-Cy3 (red). Cells were labeled for giantin (green, left panels) and EEA1 (blue, right panels). Nuclei were labeled with Hoechst (blue, left panels). Arrows indicate spots of STxB colocalized with EEA1, dotted lines indicate areas of zoomed panels. Scale bar = 10 μm. Representative images from 6 independent experiments (Giantin colocalisation) and 3 independent experiments (EEA1 colocalisation). ( b ) Quantification of STxB colocalization with Golgi area defined by giantin. Graph represents mean +/- SD for 46 (scrambled), 35 (Retro-2), 39 (siSec16A), 42 (Retro-2+siSec16A) cells from three independent experiments. Each point represents an individual cell. One-way ANOVA was performed overall (P < 0.0001) with Dunnett’s multiple comparison test to compare Scrambled to treated conditions (*** P = 0.0002, ** P = 0.0021). ( c ) Quantification of number of EEA1 spots positive for STxB. Graph represents mean +/- SD for a minimum of 40 cells from 3 independent experiments. One-way ANOVA was performed overall (P < 0.0001) with Dunnett’s multiple comparison test to compare Scrambled to treated conditions (*** P < 0.0001). ( d ) Intoxication of HeLa cells with SLT1 in the indicated conditions. HeLa cells were transfected with either scrambled siRNA, or siRNAs against Sec16A. Where indicated, cells were incubated 30 min with 25 μM Retro-2 or vehicle (0.05% DMSO, “control”), before the addition of SLT1 for 1 h at the indicated concentrations. Medium was removed and replaced for 1 h with Ca 2+ and Mg 2+ -supplemented PBS containing [ 35 S]-methionine. Each data point represents the mean of a representative experiment, from 2 independent experiments. ( e ) HeLa cells were transfected with siSec16A and where indicated treated for 30 min with 25 μM Retro-2 or DMSO control. Cells were labeled for TGN46 (green) and Syn5 (red), and nuclei labeled with Hoechst (blue). Representative images from 3 independent experiments. ( f ) Quantification of Syn5 at Golgi area as defined by TGN46. Graph represents mean +/- SD for 48 (Scrambled), 39 (Retro-2), 41 (siSec16A), 43 (Retro-2 + siSec16A) cells from 3 independent experiments. Each point represents an individual cell. One-way ANOVA was performed overall (P < 0.0001) with Dunnett’s multiple comparisons test to compare Scrambled to treated conditions (*** P < 0.0001).

Article Snippet: Antibodies against the following antigens were purchased from the indicated sources: Syn5 (Synaptic Systems, Cat#110053), GFP, giantin (institutional antibody platform at Curie Institute), TGN46 (BioRad, Cat#AHP500GT) GS27, GS28 (both BD Biosciences; Cat#611034, and Cat#611184), Sec16A (Proteintech Europe, Cat#20025-1-AP).

Techniques: Transfection, Incubation, Labeling, Comparison, Control

Journal: Nature chemical biology

Article Title: Functional Dissection of the Retrograde Shiga Toxin Trafficking Inhibitor Retro-2

doi: 10.1038/s41589-020-0474-4

Figure Lengend Snippet: ( a ) Retrograde trafficking of STxB in scrambled (-) or siGPP130 (+) treated cells. Where indicated, cells were transfected with siRNA resistant wild-type GPP130 1-108 (WT), or the KR/AA-mutant of GPP130 1-108 . Quantification of STxB-Cy3 labeling in the Golgi area in the indicated conditions. Graph shows mean +/- SD for 52 (control), 47 (siGPP130), 39 (WT rescue), 29 (KR, AA rescue) cells from 2 out of 3 independent experiments. Each point represents a cell. One-way ANOVA was performed overall (P < 0.0001) with Sidak’s multiple comparisons test. NS P = 0.8903, *** P < 0.0001. ( b ) Retrieval of GPP130 from endosomes. Gene-edited cells lacking GPP130 were transfected with either HA-GPP130 1-108 (WT) or the KR/AA-GPP130 mutant construct. Cells were then harvested before treatment (pre-treatment), treated with monensin for 1 h to redistribute GPP130 to endosomes, or monensin-treated and then subjected to a 3 h washout. Cells were labeled with GPP130 (green) and giantin (red). Representative immunofluorescence images are shown from 9 independent experiments. Dotted lines indicate nuclei, dashed lines show areas of zooms. Scale bars = 10 μm. ( c ) Quantification of number of cells with GPP130 primarily Golgi-localized, a mix of Golgi- and endosome-localized, or primarily endosome-localized, in washout conditions for WT and KR-AA transfected cells in (b). Graph shows mean +/- SD of ~50 cells from 9 independent experiments. Two-way ANOVA was performed overall (P < 0.0001) with Sidak’s multiple comparisons test. Golgi: ** P = 0.0044, Both: NS P = 0.9925, Endosomes ** P = 0.0027. ( d ) Redistribution of GPP130 in Retro-2, siSec16A, or siSar1A/B conditions. Confocal microscopy was performed for GPP130 (red), Giantin (green), EEA1 (blue in right panels) and Hoechst (blue in left panels). Dotted lines indicate zoomed areas. Arrows indicate GPP130 spots colocalizing with EEA1-positive vesicles. Scale bars = 10 μm. Representative images from 3 independent experiments. ( e ) Quantification of GPP130 in Golgi area as defined by giantin. Graph shows mean +/- SD for 31 (Scrambled), 29 (Retro-2), 27 (siSec16A), 29 (siSar1A/B) cells from 2 out of 3 independent experiments. Each point represents one cell. One-way ANOVA performed overall (P < 0.0001) with Dunnett’s multiple comparisons test comparing all conditions to scrambled, Retro-2: ** P = 0.0044, siSec16A/siSar1A/B: *** P < 0.0001. ( f ) Quantification of GPP130 in EEA1 positive structures. Graph shows mean +/- SD for 35 cells per condition from 2 out of 3 independent experiments. Each point represents a cell. One-way ANOVA performed overall (P < 0.0002) with Dunnett’s multiple comparisons test comparing all conditions to scrambled, Retro-2: * P = 0.048, siSec16A: *** P < 0.0001, siSar1A/B NS P = 0.3654. ( g ) Schematic model of Retro-2’s mode of action. (i) Under untreated conditions, Syn5 cycles in a Sec16A-dependent manner between Golgi and ER. (ii) GPP130 cycles between Golgi and plasma membrane. (iii) In this study, we show that GPP130 and Syn5 interact directly, and that this interaction is involved in Shiga toxin trafficking from endosomes to the Golgi. (iv) Retro-2 binds to Sec16A, reducing anterograde Syn5 trafficking, thereby leading to its relocalization to the ER. (v) Syn5 relocalization prevents its interaction with GPP130 and results in the inhibition of retrograde Shiga toxin trafficking.

Article Snippet: Antibodies against the following antigens were purchased from the indicated sources: Syn5 (Synaptic Systems, Cat#110053), GFP, giantin (institutional antibody platform at Curie Institute), TGN46 (BioRad, Cat#AHP500GT) GS27, GS28 (both BD Biosciences; Cat#611034, and Cat#611184), Sec16A (Proteintech Europe, Cat#20025-1-AP).

Techniques: Transfection, Mutagenesis, Labeling, Control, Construct, Immunofluorescence, Confocal Microscopy, Clinical Proteomics, Membrane, Inhibition

Journal: PLoS ONE

Article Title: Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport

doi: 10.1371/journal.pone.0157227

Figure Lengend Snippet: (A) NRK cells transfected with the indicated siRNA were labeled with anti-ALG-2, anti-sec31A, or anti-sec16A antibodies, a cy3-conjugated secondary antibody, and dapi. Maximum intensity projections from deconvolved widefield z-stacks of the ERES proteins are displayed in grayscale merged with the corresponding dapi panel. (B) Quantitation of 4–5 fields of ~10 random cells per field, for each condition. A consistent intensity threshold was used to automatically capture and count bright cytoplasmic objects. A T-test between the siControl and siPeflin group was performed for each labeled protein: ALG-2, N = 85, p = 0.0003; Sec31A, N = 79, p = 0.0001; Sec16A, N = 100, p = 0.866.

Article Snippet: Rabbit anti-sec16A (product NB100-1799) was from Novus Biologicals.

Techniques: Transfection, Labeling, Quantitation Assay

Journal: bioRxiv

Article Title: Multi-omics analysis of TNBC organoids identifies phosphorylation of the membrane trafficking machinery as key event associated with FER-mediated invasion

doi: 10.64898/2025.12.18.695070

Figure Lengend Snippet: (A) Western blot of protein extracts from MM231 SEC16A-iKD cells with or without DOX treatment, blotted with anti-SEC16A and anti-Vinculin used as loading control. Numbers on the bottom indicate the fold-changes in SEC16A levels normalised to loading control levels for each experimental condition (B) Confocal images of MM231 SEC16A-iKD cells untreated or treated with DOX, stained with DAPI (blue) and anti-SEC16A (grey). Scale bars indicate 15 µm. (C) (Left panels) Phase contrast (upper panels) or confocal images (lower panels) of MM231 SEC16A-iKD #1 spheroids in Matrigel untreated or treated with DOX. (Lower panels) cells were stained with DAPI (blue) and Phalloidin (magenta). Scale bars, 50 μm. (Right panel) Quantification of the invasiveness of MM231 SEC16A-iKD #1 spheroids untreated or treated with DOX. Statistical significance was calculated using unpaired t-test ****p<0.0001. (D) Western blot on protein extracts from TNBC PDXOs SEC16A-iKD untreated or treated with DOX, cultured in invasive conditions and blotted with anti-SEC16A and anti-HSC70 used as a loading control. Numbers on the bottom indicate the fold-changes in SEC16A levels normalised to HSC70 levels for each experimental condition. (E) (Left panels) DIC images of TNBC PDXOs SEC16A-iKD cultured in invasive conditions, untreated or treated without DOX. (Right panel) Quantification of the invasiveness of TNBC PDXOs SEC16A-iKD cultured in invasive condition, untreated or treated without DOX. Data are from three biological replicates and include more than 30 PDXOs per condition and per replicate. Statistical significance was calculated using unpaired t-test. ∗∗∗∗p<0.0001. (F) (Left panels) Confocal images of MM231 SEC16A-iKD cells untreated or treated with DOX, stained with DAPI (blue), Phalloidin (magenta), and Paxillin (grey). Scale bars indicate 15 µm. (Right panel) Quantification of the number of FAs per MM231 SEC16A-iKD cells treated with DOX (n=71) or without DOX (n=64). Data are from three biological replicates with at least three fields analysed per condition and per replicate. Each dot represents one cell. Statistical significance was calculated using unpaired t-test ****p<0.0001. (G) (Left panels) Confocal images of MM231 SEC16A-iKD cells untreated or treated without DOX, stained with DAPI (blue), Phalloidin (magenta) and anti-Rab4 (grey). Scale bars indicate 15 µm. (Right panel) Quantification of the number of Rab4 vesicles in MM231 SEC16A-iKD cells treated with DOX (n=336) or without DOX (n=496). Data are from three biological replicates, with at least three fields analysed per condition and per replicate. Each dot represents one field. Scale bars indicate 15 µm. Error bars indicate standard deviation. Statistical significance was calculated using unpaired t-test. ****p<0.0001.

Article Snippet: Anti-SEC16A primary antibodies (HPA005684, Protein Atlas Antibodies, 1:100) were manually applied and incubated at 37 °C for 32 minutes.

Techniques: Western Blot, Control, Staining, Cell Culture, Standard Deviation

Journal: bioRxiv

Article Title: Multi-omics analysis of TNBC organoids identifies phosphorylation of the membrane trafficking machinery as key event associated with FER-mediated invasion

doi: 10.64898/2025.12.18.695070

Figure Lengend Snippet: (A) MS/MS spectrum of the SEC16A phosphopeptide FTGS[pho]FDDDPDPHRDPYGEEVDR (4+), showing phosphorylation at Ser4, corresponding to Ser1327 in the full-length protein. Labelled peaks correspond to the main fragment ions observed in the spectrum. Identified b- and y-series ions, together with the precursor ion, confirm the peptide sequence. The b- ions identified support the presence and location of the phosphate group. The site was identified with a localization probability of 0.92 and a PEP score of 0.0085. (B) Confocal images of MM231 SEC16A iKD cells expressing GFP-SEC16A-WT (cyan blue), or GFP-SEC16A-S1327A (cyan blue) or GFP-SEC16A-S1327D (cyan blue), treated with DOX and stained with DAPI (blue) and anti-SEC16A (magenta). Scale bars represent 15μm. (C) (Left panels) Confocal images of MM231 SEC16A iKD cells expressing GFP-SEC16A-WT, or GFP-SEC16A-S1327A or GFP-SEC16A-S1327D, treated with DOX and stained with DAPI (blue), anti-SEC16A (magenta) and anti-Rab4 (grey). Scale bars represent 15μm. (Right panel) Quantification of the number of Rab4 vesicles in MM231 SEC16A iKD cells expressing GFP-SEC16A-WT (n=76) or GFP-SEC16A-S1327A (n=93) or GFP-SEC16A-S1327D (n=85). Data are from three biological replicates with at least three fields analysed per condition and per replicate. Each dot represents one cell. ****p<0.0001 between cells expressing WT SEC16A and S1327A SEC16A. ***p=0.0004 between cells expressing S1327A SEC16A and S1327D SEC16A. ns indicates non-significant between cells reconstituted with WT SEC16A and S1327D SEC16A (p=0.9291). Statistical significance was calculated using one-way ANOVA. (D) (Left panels) Confocal images of MM231 SEC16A iKD cells expressing GFP-SEC16A-WT (cyan blue), or GFP-SEC16A-S1327A (cyan blue) or GFP-SEC16A-S1327D (cyan blue), treated with DOX and stained with DAPI (blue) and anti-Paxillin (grey). Scale bars represent 15 μm. (Right panel) Quantification of the number of FAs in MM231 SEC16A iKD cells expressing GFP-SEC16A-WT (n=65) or GFP-SEC16A-S1327A (n=78) or GFP-SEC16A-S1327D (n=59). Data are from three biological replicates with at least three fields analysed per condition and per replicate. Each dot represents one cell. ****p<0.0001 between cells expressing WT SEC16A and S1327A SEC16A and between cells expressing S1327A SEC16A and S1327D SEC16A. ns indicates non-significant (p=0.4673) between cells expressing WT SEC16A and S1327D SEC16A. Statistical significance was calculated using one-way ANOVA. (E) (Left panel) Aminoacid sequence of the region comprising amino-acids 1307-1378. Phosphorylatable residues are highlighted: serine (magenta), threonine (green), or tyrosine (blue). Purple arrow indicates Ser1327. (Right panel) Ratios of serine (magenta), threonine (green), or tyrosine (blue) residues in the region comprising amino-acids 1307-1378.

Article Snippet: Anti-SEC16A primary antibodies (HPA005684, Protein Atlas Antibodies, 1:100) were manually applied and incubated at 37 °C for 32 minutes.

Techniques: Tandem Mass Spectroscopy, Phospho-proteomics, Sequencing, Expressing, Staining

Journal: bioRxiv

Article Title: Multi-omics analysis of TNBC organoids identifies phosphorylation of the membrane trafficking machinery as key event associated with FER-mediated invasion

doi: 10.64898/2025.12.18.695070

Figure Lengend Snippet: (A) Western blot on protein extracts from MM231 FER-iKD cells untreated or treated with DOX, blotted with anti-SEC16A or anti-HSC70 used as a loading control. Numbers on the bottom indicate the fold-changes in SEC16A levels normalised to HSC70 levels for each experimental condition. (B) (Left panels) Confocal images of MM231 FER-iKD cells untreated or treated with DOX, stained with DAPI (blue) and anti-SEC16A (grey). Scale bars represent 15 µm. (Right panel) Quantification of the shortest distance between SEC16A signals and the nucleus in MM231 FER-iKD cells treated with DOX (n=113) or without DOX (n=188). Data are from three biological replicates and include at least three fields analysed per condition and per replicate. Each dot represents one field. Error bars indicate standard deviation. Statistical significance was calculated using unpaired t-test. ****p<0.0001. (C and D) Multispectral immunofluorescence imaging of a TNBC PDXO #1 tumour xenograft invading on CAM. Scale bars represent 30 µm. Tissue samples were stained with anti-SEC16A (magenta), anti-FER (yellow), anti-Rab4 (orange), anti-Integrin β1 (cyan blue), anti-E-cad (red) and DAPI (blue). (D) Inset images highlight the levels of FER and SEC16A in leader invading cells. White arrows indicate leader cells. Scale bars represent 10 µm. (E) IHC images of TNBC clinical specimens, stained for SEC16A and FER. Scale bars: 50 µm. (F) Correlation between SEC16A and FER expression in Luminal A, Luminal B, HER2 and TNBC/basal breast cancer. Statistical significance estimated by χ 2-test. P-values are indicated. Statistically significant p-values are indicated in bold. (F) Model by which phosphorylated SEC16A acts, downstream of FER, to coordinate the formation of Rab4-positive endosomes and focal adhesions and sustain invasion in TNBC. In TNBC, high levels of FER correlate with low SEC16A levels. Still, a certain amount of SEC16A protein is necessary to sustain FER-dependent endosomal recycling and invasion of integrins in TNBC.

Article Snippet: Anti-SEC16A primary antibodies (HPA005684, Protein Atlas Antibodies, 1:100) were manually applied and incubated at 37 °C for 32 minutes.

Techniques: Western Blot, Control, Staining, Standard Deviation, Immunofluorescence, Imaging, Expressing

Journal: Annals of the Rheumatic Diseases

Article Title: Private rare deletions in SEC16A and MAMDC4 may represent novel pathogenic variants in familial axial spondyloarthritis

doi: 10.1136/annrheumdis-2014-206484

Figure Lengend Snippet: Clinical information, genotype data ( HLA-B27 , SEC16A and MAMDC4 ) of the large, well-characterised, multigenerational family with numerous individuals clinically diagnosed with and without AxSpA

Article Snippet: Real-time PCR was performed using TaqMan Gene Expression Assays for SEC16A (Hs_00389570_m1) and GAPDH (Hs_99999905_m1) from Life Technologies.

Techniques: Biomarker Discovery

Journal: Annals of the Rheumatic Diseases

Article Title: Private rare deletions in SEC16A and MAMDC4 may represent novel pathogenic variants in familial axial spondyloarthritis

doi: 10.1136/annrheumdis-2014-206484

Figure Lengend Snippet: Deletions located within SEC16A and MAMDC4 . (A) Full exome sequencing analysis revealed a 9 base pair deletion located within SEC16A and a 20 base pair deletion located within MAMDC4 that segregated only within affected family members. Both deletions were confirmed using bidirectional Sanger sequencing of the proband (II-1). (B) The top panel is a representative chromatogram illustrating a portion of the wild-type sequence of SEC16A exon 3. The lower panel is a representative chromatogram illustrating a portion of SEC16A exon 3 with the in-frame deletion indicated by an arrow. (C) The top panel is a representative chromatogram illustrating a portion of the wild-type sequence of MAMDC4 exon 5. The lower panel is a representative chromatogram illustrating a portion of MAMDC4 exon 5 with the out-of-frame deletion indicated by an arrow.

Article Snippet: Real-time PCR was performed using TaqMan Gene Expression Assays for SEC16A (Hs_00389570_m1) and GAPDH (Hs_99999905_m1) from Life Technologies.

Techniques: Sequencing

Journal: Annals of the Rheumatic Diseases

Article Title: Private rare deletions in SEC16A and MAMDC4 may represent novel pathogenic variants in familial axial spondyloarthritis

doi: 10.1136/annrheumdis-2014-206484

Figure Lengend Snippet: Linkage analysis was performed using VCFtools to estimate the degree of linkage between the SEC16A and MAMDC4 loci in the family and the general population. Analysis revealed that there is very strong linkage disequilibrium (LD) (r 2 =1; D′=1) between the SEC16A and MAMDC4 loci within the nuclear subfamily. The X and Y axes are genomic positions on chromosome 9, the deletions in SEC16A and MAMDC4 are located at positions 139370954 and 139748276, respectively. White represents a score of 0 and black represents a score of 1. Linkage disequilibrium cluster plots illustrating (A) r 2 values and (B) the D′ values for the nuclear family. In contrast, (C) SEC16A and (D) MAMDC4 loci occur in distinctly separate LD blocks in the general population.

Article Snippet: Real-time PCR was performed using TaqMan Gene Expression Assays for SEC16A (Hs_00389570_m1) and GAPDH (Hs_99999905_m1) from Life Technologies.

Techniques:

Journal: Annals of the Rheumatic Diseases

Article Title: Private rare deletions in SEC16A and MAMDC4 may represent novel pathogenic variants in familial axial spondyloarthritis

doi: 10.1136/annrheumdis-2014-206484

Figure Lengend Snippet: SEC16A gene and SEC16A protein expression is not altered among individuals with the deletion mutation. (A) The relative expression of SEC16A mRNA was determined by real-time quantitative PCR (qPCR) analysis of patient-derived B-cell lines. SEC16A relative expression was compared between family members with wild-type (Wt) and heterozygous (Het) genotypes for the SEC16A and MAMDC4 deletion. Results are expressed as mean±SEM and analysed by t test. (B) Sec16A protein expression of RIPA lysates from patient-derived B-cell lines of family members. Blots are representative of three separate experiments. Densitometric analysis was performed to determine the level of SEC16A expression relative to tubulin as a loading control. (C) Comparison of protein expression between family members with Wt and Het genotypes for the SEC16A deletion. Results are expressed as mean±SEM and analysed by t test.

Article Snippet: Real-time PCR was performed using TaqMan Gene Expression Assays for SEC16A (Hs_00389570_m1) and GAPDH (Hs_99999905_m1) from Life Technologies.

Techniques: Expressing, Mutagenesis, Real-time Polymerase Chain Reaction, Derivative Assay, Control, Comparison

Journal: Annals of the Rheumatic Diseases

Article Title: Private rare deletions in SEC16A and MAMDC4 may represent novel pathogenic variants in familial axial spondyloarthritis

doi: 10.1136/annrheumdis-2014-206484

Figure Lengend Snippet: Circular dichroism (CD) spectroscopy performed on the 40 amino acid peptide and the complete N-terminus peptide of Sec16A. (A) CD spectroscopy of the 40 amino acid peptides demonstrated that both the Sec16A wild-type (o; black) and mutant (Δ; green) peptides have a secondary structure that is disordered. The wild-type peptide (□; blue) in the presence of trifluoroethanol (TFE) adopted a distinct secondary structure (broad peak from 230 to 200 nm and the disappearance of the negative peak at 195 nm). The mutant peptide (+; red) in the presence of TFE failed to adopt a distinct secondary structure. (B) CD spectroscopy of the complete N-terminus also demonstrated that both the Sec16A wild-type (o; black) and mutant (Δ; green) peptides have a secondary structure that is disordered. The wild-type peptide (□; blue) in the presence of TFE adopted a distinct secondary structure, whereas the mutant peptide (+; red) in the presence of TFE had a spectra similar in shape to the wild-type peptide, but exhibited a decreased signal amplitude. Each CD spectroscopy graph is representative of the average of 30 spectra.

Article Snippet: Real-time PCR was performed using TaqMan Gene Expression Assays for SEC16A (Hs_00389570_m1) and GAPDH (Hs_99999905_m1) from Life Technologies.

Techniques: Circular Dichroism, Mutagenesis

Journal: Frontiers in Immunology

Article Title: NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses

doi: 10.3389/fimmu.2019.00958

Figure Lengend Snippet: SEC16A forms a complex with PI31 and TBK1. (A) Immunoblot analysis representing immunoprecipitation of endogenous PI31 in DCs following MDP/PAM 3 CSK 4 stimulation for LC-MS/MS analysis. (B) List of proteins from LC/MS analysis with highest confidence score and peptide counts. (C) Relative abundance protein for SEC16A. Data represent mean ± s.e.m; ( n = 2) * P < 0.05; one-way ANOVA (Tukey's multiple comparison test). (D) DCs and (E) THP1 cells stimulated with MDP/PAM 3 CSK 4 . Cell lysates were immunoprecipitated with PI31 antibodies and probed with anti-SEC16A. Data are representative of three independent experiments. Densitometric analysis of SEC16A band intensities normalized with αctin ( n = 3). Data represent the mean ± s.e.m. ( n = 3); * P < 0.05 Student's t test. (F,G) Images show confocal analysis of THP-1 cells transfected with GFP-PI31 (green) for 24 h, and stimulated with MDP/PAM 3 CSK 4 for 30 min and stained with antibody to SEC16A (red) and DAPI (blue). Pearson's coefficient for PI31 and SEC16A co-localization in control or MDP/PAM 3 CSK 4 stimulated cells. Data represent means ± s.e.m. ( n = 20), Three independent experiments with at least five images analyzed per experiment); **** P < 0.0001 Student's t -test. (H) THP1 cells were stimulated with MDP/PAM 3 CSK 4 for 30 min. Cell lysates were immunoprecipitated with anti-TBK1 antibodies and probed with anti-SEC16A, anti-TBK1 and anti-TANK antibodies. Data represent three independent experiments. Densitometric analysis of TBK1 band intensity normalized with αctin ( n = 3). Data represent the mean ± s.e.m. ( n = 3); n.s., non-significant. Student's t -test.

Article Snippet: The antibodies used in the study include: rabbit anti-human PI31 (Atlas Antibodies, HPA041122), rabbit anti-mouse PI31 (Abcam, ab187200); rabbit anti-human P38 (9212), rabbit anti-mouse/human actin (12620), anti-human TBK1 (3504), anti-human IRF-3 (11904), rabbit anti-tubulin (5335), mouse anti-human PSMB8/LMP7 (13726), rabbit anti- human PA28α (9643), rabbit anti-human PSMD2 (25430), rabbit anti-human PSMA2 (11864), rabbit anti-human TANK (2141), rabbit anti-phospho-NF-κB p65 (3033), rabbit anti-human IκBα (9242), mouse anti-human phospho-IκBα (9246), rabbit anti-mouse ERp72 (5033), rabbit anti-mouse LAMP1 (3243), rabbit anti-FLAG (14793), rabbit anti-HA (3724), rabbit anti-GFP (2956), anti-mouse (7076), and anti-rabbit (7074) IgG HRP-linked secondary antibodies were all from Cell Signaling; rabbit-anti-human SEC16A (Abcam, ab70722), rabbit anti-mouse SEC16A (Holzel diagnostika, KIAA0310), mouse anti-human PSMA2 (Enzo, BML-PW8105-0100), mouse anti-human PSMA4 (Enzo, BML-PW8120).

Techniques: Western Blot, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Transfection, Staining

Journal: Frontiers in Immunology

Article Title: NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses

doi: 10.3389/fimmu.2019.00958

Figure Lengend Snippet: PI31, but not SEC16A, mediates NOD2/TLR2-dependent cross-presentation. (A) PI31 expression was downregulated by transfecting BMDCs with control non-targeting and PI31 siRNAs. Immunoblots data represent three independent experiments. (B,C) 48 h post-transfection, BMDCs were incubated with MDP/PAM 3 CSK 4 in presence or absence of sOVA (0, 0.1, and 1 μg/ml) or SIINFEKL peptide for 3 h followed by co-culture with OT-I CD8 + T cells for 72 h. Cross-presentation efficiency of DCs was determined by the percentage of CD8 + CD69 + T cells using FACS. Data represent the mean ± s.e.m; ( B,C , n = 4). Data are from one experiment representative of three or more independent experiments. n.s. = non-significant; **** P < 0.0001; * P < 0.05; one-way ANOVA (Tukey's multiple comparison test) (D) SEC16A expression was downregulated by transfecting BMDCs with control non-targeting and SEC16A siRNAs. Immunoblots data represent three independent experiments. (E,F) 48 h post-transfection, BMDCs were incubated with MDP/PAM 3 CSK 4 with or without sOVA (0, 0.1, and 1 ug/ml) or SIINFEKL peptide for 3 h followed by co-culture with OT-I CD8 + T cells for 72 h. Cross-presentation was measured by calculating the percentage of CD8 − CD69 − T cells using FACS. Data represent mean ± s.e.m; ( E,F , n = 3). Data from one experiment representative of three or more independent experiments. n.s., non-significant; one-way ANOVA (Tukey's multiple comparison test).

Article Snippet: The antibodies used in the study include: rabbit anti-human PI31 (Atlas Antibodies, HPA041122), rabbit anti-mouse PI31 (Abcam, ab187200); rabbit anti-human P38 (9212), rabbit anti-mouse/human actin (12620), anti-human TBK1 (3504), anti-human IRF-3 (11904), rabbit anti-tubulin (5335), mouse anti-human PSMB8/LMP7 (13726), rabbit anti- human PA28α (9643), rabbit anti-human PSMD2 (25430), rabbit anti-human PSMA2 (11864), rabbit anti-human TANK (2141), rabbit anti-phospho-NF-κB p65 (3033), rabbit anti-human IκBα (9242), mouse anti-human phospho-IκBα (9246), rabbit anti-mouse ERp72 (5033), rabbit anti-mouse LAMP1 (3243), rabbit anti-FLAG (14793), rabbit anti-HA (3724), rabbit anti-GFP (2956), anti-mouse (7076), and anti-rabbit (7074) IgG HRP-linked secondary antibodies were all from Cell Signaling; rabbit-anti-human SEC16A (Abcam, ab70722), rabbit anti-mouse SEC16A (Holzel diagnostika, KIAA0310), mouse anti-human PSMA2 (Enzo, BML-PW8105-0100), mouse anti-human PSMA4 (Enzo, BML-PW8120).

Techniques: Expressing, Western Blot, Transfection, Incubation, Co-Culture Assay

Journal: bioRxiv

Article Title: Insulin promoted mobilization of GLUT4 from a perinuclear storage site requires RAB10

doi: 10.1101/2020.04.14.040683

Figure Lengend Snippet: A and B. Representative Airyscan confocal single plane images of (A) basal and (B) insulin-stimulated cells expressing BFP-RAB10 and HA-GLUT4-GFP, and labeled for endogenous SEC16A by IF. Serum starved cells stimulated with 1nM insulin. Inset (white, dashed boxed region) displayed below. Linescan plot is BFP-RAB10, HA-GLUT4-GFP, and SEC16A fluorescence intensity along a line (indicated by white arrow). Values normalized to each individual fluorescence maxima. Bars, 5 μm.

Article Snippet: Antibodies against Syntaxin6 (ab12370; Abcam and 2869T; Cell Signaling), TGN46 (ab16059; Abcam), LAMP1 (ab25630; Abcam), ATP7A (LS-C209614; LSBio), GM130 (610822; BD Transduction), SEC16A (KIAA0310; ProteinExpress), and Haemagglutinin (HA) tag (901503; Biolegend) were used for immunofluorescence.

Techniques: Expressing, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Insulin promoted mobilization of GLUT4 from a perinuclear storage site requires RAB10

doi: 10.1101/2020.04.14.040683

Figure Lengend Snippet: A and B. Representative Airyscan confocal single plane images of cells treated with 3μM nocodazole. Cells expressing HA-GLUT4-GFP and BFP-RAB10 and stained for endogenous SEC16A by IF. Cells under (A) basal and (B) 1nM insulin-stimulated conditions. Inset (white, dashed boxed region) displayed below. Yellow arrows indicate the same position in each image. Bars, 5 μm. C and D. Images of the average HA-GLUT4-GFP, SEC16A, and BFP-RAB10 fluorescence intensity from 5 individual fragments, centered of HA-GLUT4-GFP, resulting from nocodazole treatment from the cells in A and B respectively. Radial linescan plot of images displayed below. Values normalized to each individual fluorescence maxima. Bars, 1 μm. E. Quantification of the distance (μm) between HA-GLUT4-GFP and SEC16A fluorescence peaks in basal cells in the presence and absence of nocodazole treatment. Values are distances between peaks ± SEM. Distance measured for 3 separate sets of peaks per cell. N=2 assays, 7-8 cells per assay. F. Representative Airyscan confocal single plane images of cells treated with 3μM nocodazole in the presence of 200μM BCS. Cells expressing HA-GLUT4-GFP and stained for endogenous ATP7A and Syntaxin6 by IF. Inset (white, dashed boxed region) displayed below. Yellow arrows indicate the same position in each image. Bars, 5 μm. G. Quantification of the fraction of BFP-RAB10 in the perinuclear region of basal and 1nM insulin-stimulated cells ± addition of siRNA targeting SEC16A. N=7 assays ± SEM. Dashed line connects data from individual assays. *, p<0.05, two-tailed unpaired t-test, nonnormalized raw data.

Article Snippet: Antibodies against Syntaxin6 (ab12370; Abcam and 2869T; Cell Signaling), TGN46 (ab16059; Abcam), LAMP1 (ab25630; Abcam), ATP7A (LS-C209614; LSBio), GM130 (610822; BD Transduction), SEC16A (KIAA0310; ProteinExpress), and Haemagglutinin (HA) tag (901503; Biolegend) were used for immunofluorescence.

Techniques: Expressing, Staining, IF-cells, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Insulin promoted mobilization of GLUT4 from a perinuclear storage site requires RAB10

doi: 10.1101/2020.04.14.040683

Figure Lengend Snippet: In 3T3-L1 adipocytes the biogenesis of GLUT4-containing vesicles (IRVs), copper transporter ATP7A-containing vesicles, and vesicles containing lysosomal enzymes occurs at a regulated domain of the trans -Golgi network (TGN); traffic of constitutive recycling proteins through the TGN occurs at an independent domain. Mobilization of ATP7A from the TGN is promoted by copper stimulation. The diversion of vesicles containing lysosomal enzymes away from traffic from the PM is mediated by the AP1 clathrin adaptor. The exocytosis of GLUT4 to the PM is accelerated by insulin. Insulin accelerates the recruitment, docking, and fusion of GLUT4-containing insulin responsive vesicles (IRVs) with the PM. Insulin also promotes the mobilization of GLUT4 from the perinuclear TGN, replenishing the IRV pool. This is important because GLUT4 in the PM is rapidly trafficked back to the TGN via the endosomal pathway. Mobilization of GLUT4 from the perinuclear region is regulated by TBC1D4, and insulin-stimulated acceleration of GLUT4 mobilization requires RAB10. Inset, SEC16A-labeled structures reside adjacent to GLUT4-containing membranes, and SEC16A organizes RAB10 at the perinuclear region.

Article Snippet: Antibodies against Syntaxin6 (ab12370; Abcam and 2869T; Cell Signaling), TGN46 (ab16059; Abcam), LAMP1 (ab25630; Abcam), ATP7A (LS-C209614; LSBio), GM130 (610822; BD Transduction), SEC16A (KIAA0310; ProteinExpress), and Haemagglutinin (HA) tag (901503; Biolegend) were used for immunofluorescence.

Techniques: Labeling