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: bioRxiv

Article Title: EMT activates ER-to-Golgi trafficking through upregulation of REEP2 to promote lung cancer progression

doi: 10.1101/2025.11.12.688098

Figure Lengend Snippet: (A) Schematic illustration of a secretory pathway model. ZEB1 upregulates REEP2 to promote ERES (SEC16, SEC24) transportation to the Golgi (GM130). (B) Confocal micrographs of cells co-stained with anti-SEC16A (green) and anti-GM130 (red) antibodies. DAPI (blue). Scale bar: 5 μm. The scatter plots quantify Golgi-localized SEC16A per cell (dot) based on % of total SEC16A that co-localizes with Golgi (GM130 channel) (n = 15 cells per group). (C-E) Confocal micrographs of cells co-transfected with SEC24D-mCherry (green) and Golgi-pmTurquoise2 (red). DAPI (blue). Scale bar: 5 μm. The scatter plots quantify Golgi-localized SEC24D per cell (dot) based on % of total SEC24D that co-localizes with Golgi (n = 15 cells per group) in HCC827 cells with ectopic ZEB1-expression (C), and with REEP2 depletion (D) or REEP2-GFP transfection (E). (F) Confocal micrographs of cells co-transfected with REEP2-GFP (green), SEC24D-mCherry (red) and Golgi-pmTurquoise2 (blue). Scale bar: 5 μm, 1 μm (inset). The scatter plots quantify Golgi-localized SEC24D per cell (dot) based on % of total SEC24D that co-localizes with Golgi (n = 15 cells per group) in HCC827 cells with REEP2-GFP transfection. Results represent means ± SEM. P values were determined using two-tailed Student’s t-test.

Article Snippet: We purchased fetal bovine serum (FBS), live-cell imaging solution, phosphate-buffered saline (PBS), RPMI-1640, Trypsin-EDTA (0.25%), Alexa Fluor-tagged secondary antibodies, paraformaldehyde, bovine serum albumin (BSA), DAPI and Triton X-100, High-Capacity cDNA Reverse Transcription Kit, PowerUpTM SYBRTM Green Master Mix, and protease/phosphatase inhibitor cocktail from Thermo Fisher Scientific; Doxycycline (#D9891) from MilliporeSigma; Bronchial Epithelial Cell Growth Medium BulletKit from Lonza; jetPRIME transfection reagent from Polyplus Transfection; Transwell and Matrigel-coated Boyden chambers from BD Biosciences; Glass-bottom dishes and multiwell plates from MatTek; 10X Cell lysis buffer from Cell Signaling Technologies; 2X cell lysis buffer (#1610737) from Bio-Rad; CCK-8 (#K1018) from APExBIO; RNeasy Plus Mini Kit (#74136), and AllStars Negative Control siRNA (#1027281) from QIAGEN; Pre-miRTM miRNA Precursor Negative Control (miR-NC) (#AM17110); human miR-183a mimics (#PM12303) and human miR-193 mimics (#PM11786) from Thermo Fisher Scientific; human REEP2 siRNA #2 (#J-021201-18), and #4 (J-021201-20) from Horizon; primary antibodies against β-actin (#4967), ZEB1 (#3396) from Cell Signaling Technologies, REEP2 (#Ab191410) from Abcam, SEC16A (#20025-1-AP) from Proteintech, VSV-G (#EB0012) from Kerafast; Alexa Fluor 555 Mouse anti-GM130 (#560066) from BD Biosciences; REEP2-GFP (#RC202507L4) from Origene; SEC24D-mCherry (#32677), Golgi-pmTurquoise2 (#36205), mCherry-Golgi (#55052) and CD-MPR-GFP-RUSH (#202797) from Addgene; 3’-UTR luciferase reporters of REEP2 (#38786081) from Applied Biological Materials; pCI-Neo Renilla luciferase reporter (Basic) (#E1841) and pGL3 Basic Firefly luciferase control reporter (#E1751) from Promega; All-in-One miRNA qRT-PCR Detection Kit 2.0 (#QP115) from GeneCopoeia.

Techniques: Staining, Transfection, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: EMT activates ER-to-Golgi trafficking through upregulation of REEP2 to promote lung cancer progression

doi: 10.1101/2025.11.12.688098

Figure Lengend Snippet: (A-C) Confocal micrographs of cells co-stained with anti-SEC16A (green) and anti-GM130 (red) antibodies. DAPI (blue). Scale bar: 5 μm. The scatter plots quantify Golgi-localized SEC16A per cell (dot) based on % of total SEC16A that co-localizes with Golgi (GM130 channel) (n = 15 cells per group) in murine epithelial cells (393P) with ectopic ZEB1-expression (A), in murine mesenchymal cells (344SQ) with ZEB1 depletion (B), or in human mesenchymal cells (H1299) with ZEB1 or REEP2 depletions (C). (D) WB analysis of REEP2 protein expression levels in HCC827_ZEB1 cells transfected with control siRNA (siCTL) or REEP2 siRNA (siREEP2). β-actin loading control. (E) WB analysis of REEP2 protein expression levels in HCC827 cells co-transfected with empty vector (Vec) or ZEB1, with REEP2-GFP. β-actin loading control. (F) Confocal micrographs of cells co-transfected with REEP2-GFP (green) and SEC24D-mCherry (red). Scale bar: 5 μm. The scatter plots quantify SEC24D-localized REEP2 per cell (dot) based on % of total REEP2 that co-localizes with SEC24D (n = 15 cells per group) in HCC827 cells with ectopic ZEB1-expression. Results represent means ± SEM. P values were determined using two-tailed Student’s t-test.

Article Snippet: We purchased fetal bovine serum (FBS), live-cell imaging solution, phosphate-buffered saline (PBS), RPMI-1640, Trypsin-EDTA (0.25%), Alexa Fluor-tagged secondary antibodies, paraformaldehyde, bovine serum albumin (BSA), DAPI and Triton X-100, High-Capacity cDNA Reverse Transcription Kit, PowerUpTM SYBRTM Green Master Mix, and protease/phosphatase inhibitor cocktail from Thermo Fisher Scientific; Doxycycline (#D9891) from MilliporeSigma; Bronchial Epithelial Cell Growth Medium BulletKit from Lonza; jetPRIME transfection reagent from Polyplus Transfection; Transwell and Matrigel-coated Boyden chambers from BD Biosciences; Glass-bottom dishes and multiwell plates from MatTek; 10X Cell lysis buffer from Cell Signaling Technologies; 2X cell lysis buffer (#1610737) from Bio-Rad; CCK-8 (#K1018) from APExBIO; RNeasy Plus Mini Kit (#74136), and AllStars Negative Control siRNA (#1027281) from QIAGEN; Pre-miRTM miRNA Precursor Negative Control (miR-NC) (#AM17110); human miR-183a mimics (#PM12303) and human miR-193 mimics (#PM11786) from Thermo Fisher Scientific; human REEP2 siRNA #2 (#J-021201-18), and #4 (J-021201-20) from Horizon; primary antibodies against β-actin (#4967), ZEB1 (#3396) from Cell Signaling Technologies, REEP2 (#Ab191410) from Abcam, SEC16A (#20025-1-AP) from Proteintech, VSV-G (#EB0012) from Kerafast; Alexa Fluor 555 Mouse anti-GM130 (#560066) from BD Biosciences; REEP2-GFP (#RC202507L4) from Origene; SEC24D-mCherry (#32677), Golgi-pmTurquoise2 (#36205), mCherry-Golgi (#55052) and CD-MPR-GFP-RUSH (#202797) from Addgene; 3’-UTR luciferase reporters of REEP2 (#38786081) from Applied Biological Materials; pCI-Neo Renilla luciferase reporter (Basic) (#E1841) and pGL3 Basic Firefly luciferase control reporter (#E1751) from Promega; All-in-One miRNA qRT-PCR Detection Kit 2.0 (#QP115) from GeneCopoeia.

Techniques: Staining, Expressing, Transfection, Control, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: EMT activates ER-to-Golgi trafficking through upregulation of REEP2 to promote lung cancer progression

doi: 10.1101/2025.11.12.688098

Figure Lengend Snippet: (A) Schematic illustration of the retention using selective hooks (RUSH) system. The cells were co-transfected with the reporter protein (CD-MPR), which is fused to the streptavidin-binding peptide (SBP) and GFP, and a second protein with a C-terminal ER retention signal (Lys-Asp-Glu-Leu; KDEL) fusing with streptavidin as a hook. The interaction of CD-MPR with the hook protein retains CD-MPR in the ER compartment and biotin administration releases the CD-MPR-GFP reporter from the ER and transfers it to the Golgi. (B) Schematic of VSV-G assay. The cells were transfected with vectors that express an EGFP-tagged temperature-sensitive mutant VSV-G (EGFP-VSV-G [ts045]) that is transported from the ER to the plasma membrane via the Golgi. At designated time points after switching cells to temperatures that cause VSV-G accumulation (40°C) and release (32°C) from the ER, cells were fixed and exofacial VSV-G was detected in non-permeabilized cells by staining with anti-VSV-G monoclonal antibody. The VSV-G trafficking to the plasma membrane is determined by the ratio of exofacial (surface) VSV-G fluorescence signal to the EGFP signal intensity. (C) Confocal micrographs of cells co-stained with anti-SEC16A (green) and anti-GM130 (red) antibodies. DAPI (blue). Scale bar: 5 μm. The scatter plots quantify Golgi-localized SEC16A per cell (dot) based on % of total SEC16A that co-localizes with Golgi (GM130 channel) (n = 15 cells per group) in H1299 cells treated with vehicle control (Veh) or 5 µM thapsigargin (Tg) for 1 h. (D) Confocal micrographs of EGFP-VSV-G-transfected cells taken 1 h after transfer to permissive temperature. Scale bar, 5 μm. The scatter plot represents the ratio of surface VSV-G to EGFP-VSV-G in each cell (dot) (n = 15 cells per group) in H1299 cells treated with vehicle control (Veh) or 5 µM thapsigargin (Tg) for 1 h. Results represent means ± SEM. P values were determined using two-tailed Student’s t-test.

Article Snippet: We purchased fetal bovine serum (FBS), live-cell imaging solution, phosphate-buffered saline (PBS), RPMI-1640, Trypsin-EDTA (0.25%), Alexa Fluor-tagged secondary antibodies, paraformaldehyde, bovine serum albumin (BSA), DAPI and Triton X-100, High-Capacity cDNA Reverse Transcription Kit, PowerUpTM SYBRTM Green Master Mix, and protease/phosphatase inhibitor cocktail from Thermo Fisher Scientific; Doxycycline (#D9891) from MilliporeSigma; Bronchial Epithelial Cell Growth Medium BulletKit from Lonza; jetPRIME transfection reagent from Polyplus Transfection; Transwell and Matrigel-coated Boyden chambers from BD Biosciences; Glass-bottom dishes and multiwell plates from MatTek; 10X Cell lysis buffer from Cell Signaling Technologies; 2X cell lysis buffer (#1610737) from Bio-Rad; CCK-8 (#K1018) from APExBIO; RNeasy Plus Mini Kit (#74136), and AllStars Negative Control siRNA (#1027281) from QIAGEN; Pre-miRTM miRNA Precursor Negative Control (miR-NC) (#AM17110); human miR-183a mimics (#PM12303) and human miR-193 mimics (#PM11786) from Thermo Fisher Scientific; human REEP2 siRNA #2 (#J-021201-18), and #4 (J-021201-20) from Horizon; primary antibodies against β-actin (#4967), ZEB1 (#3396) from Cell Signaling Technologies, REEP2 (#Ab191410) from Abcam, SEC16A (#20025-1-AP) from Proteintech, VSV-G (#EB0012) from Kerafast; Alexa Fluor 555 Mouse anti-GM130 (#560066) from BD Biosciences; REEP2-GFP (#RC202507L4) from Origene; SEC24D-mCherry (#32677), Golgi-pmTurquoise2 (#36205), mCherry-Golgi (#55052) and CD-MPR-GFP-RUSH (#202797) from Addgene; 3’-UTR luciferase reporters of REEP2 (#38786081) from Applied Biological Materials; pCI-Neo Renilla luciferase reporter (Basic) (#E1841) and pGL3 Basic Firefly luciferase control reporter (#E1751) from Promega; All-in-One miRNA qRT-PCR Detection Kit 2.0 (#QP115) from GeneCopoeia.

Techniques: Transfection, Binding Assay, Mutagenesis, Clinical Proteomics, Membrane, Staining, Fluorescence, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Multi-scale Molecular Imaging of Human Cells reveals COPI and COPII Vesicles at ER Exit Sites

doi: 10.1101/2025.07.29.667472

Figure Lengend Snippet: A. Three-colour high resolution confocal fluorescence imaging of endogenous HaloTag-Sec23A RPE1 cells dyed with HaloTag JFX650 ligand (green) and stained with antibodies directed against Sec16 (orange) and 𝛽-COP (purple). B. Quantitative data highlighting the relative distribution of COPII (HaloTag-Sec23A) and transitional ER (Sec16A). Left panel: Violin plots of the distance between centroids of endogenous HaloTag-Sec23A puncta from immunostained Sec16 and 𝛽-COP. Extracted from the three-color high resolution confocal fluorescence imaging as exampled in A. For Sec16, n = 5819, from ≥10 cells, across 3 experimental repeats. For 𝛽-COP, n = 4950, from ≥10 cells, across 4 experimental repeats. Right panel: Averaged linescans of Sec16 (orange) and HaloTag-Sec23A (green), extracted from two-colour super resolution STED imaging and fitted from 50 structures across 2 cells over 1 experimental repeat. Standard error is shown by the dotted lines. The intensity values were normalized for display purposes. C. Three-color high resolution confocal fluorescence imaging of endogenous HaloTag-Sec23A RPE1 cells dyed with HaloTag JFX650 ligand (green) and stained with antibodies directed against ERGIC-53 (cyan) and 𝛽-COP (purple). D. Two-colour super resolution STED imaging of endogenous SNAP-tag-ERGIC-53 RPE1 cells stained with antibodies directed against 𝛽-COP (purple) and SNAP-tag-ERGIC-53 dyed with JFX650-SNAP-tag ligand (cyan) Scale bar: A,C overviews: 1 µm, insets: 200 nm, D: 100 nm

Article Snippet: Immunofluorescence studies were conducted using validated antibodies directed against Sec16A (Bethyl Laboratories; A300-648A), Sec31A (BD Sciences; 612351), TFG (Novus Biologicals; NBP2-62212), ERGIC-53 (Santa Cruz Biotechnology; sc-66880), and COPB (Santa Cruz Biotechnology; sc-393615).

Techniques: Fluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: Multi-scale Molecular Imaging of Human Cells reveals COPI and COPII Vesicles at ER Exit Sites

doi: 10.1101/2025.07.29.667472

Figure Lengend Snippet: An updated model of the mammalian ERES. COPII coated vesicles bud off Sec16A-positive segments of the ER membrane in ribosome-free regions of the cytosol. COPII vesicles are clustered by TFG in the area between the transitional ER and the ERGIC. COPI vesicles bud off ERGIC vesicular-tubular membranes and are found in close proximity to COPII.

Article Snippet: Immunofluorescence studies were conducted using validated antibodies directed against Sec16A (Bethyl Laboratories; A300-648A), Sec31A (BD Sciences; 612351), TFG (Novus Biologicals; NBP2-62212), ERGIC-53 (Santa Cruz Biotechnology; sc-66880), and COPB (Santa Cruz Biotechnology; sc-393615).

Techniques: Membrane

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: Molecular Neurodegeneration

Article Title: Pathological characteristics of axons and alterations of proteomic and lipidomic profiles in midbrain dopaminergic neurodegeneration induced by WDR45-deficiency

doi: 10.1186/s13024-024-00746-4

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: SEC16A , Rabbit , IFC , 1:400 , Proteintech , 20,025–1-AP.

Techniques: Ubiquitin Proteomics