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: 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: 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: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: RAB10-interacting proteins identified by mass spectrometry

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: Activation Assay

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: SEC16A is required for GLUT4 translocation. (A) Quantification of PM to total HA-GLUT4-GFP in adipocytes transfected with siRNA targeting the noted proteins. More than 20 cells quantified per condition per assay. Mean normalized values ± SEM. (B) Epifluorescence images of HA-GLUT4-GFP in basal and 1 nM of insulin-stimulated control and SEC16A knockdown adipocytes. Total expression of HA-GLUT4-GFP is revealed by the GFP fluorescence, and PM is revealed by immunofluorescence (IF) against HA epitope in fixed, nonpermeabilized cells. (C) Quantification of PM (surface) to total HA-GLUT4-GFP in SEC16A knockdown adipocytes with or without SEC16A cDNA. Mean normalized values ± SEM. (D) SEC16A knockdown cells are binned into quartiles based on the integrated residual SEC16A expression (x axis) determined by IF of endogenous SEC16A. The mean GLUT4 surface-to-total of cells in each quartile ± SEM, normalized to the WT population mean GLUT4 surface-to-total, is plotted (y-axis). 40 cells analyzed/assay. (E) SEC16A knockdown cells binned into quartiles based on residual SEC16A expression (x axis). Shown is the mean integrated anti-SEC16A (Cy5 fluorescence) intensity of cells in each quartile ± SEM, normalized to the control population mean anti-SEC16A intensity (y axis). 40 cells were analyzed in each assay. (F) Representative epifluorescence images from D and E of HA-GLUT4-GFP–expressing cells with the highest (Q4) and lowest (Q1) expression of SEC16A. Total HA-GLUT4-GFP is revealed by the GFP fluorescence and PM by IF against the HA epitope in fixed, nonpermeabilized cells. Endogenous SEC16A revealed by IF. Red arrow notes high expression of perinuclear SEC16A. In contrast, low-expressing cells lack this perinuclear accumulation of SEC16A while maintaining diffuse background anti-SEC16A staining. (G) Representative Airyscan confocal single-plane images of HA-GLUT4-GFP–expressing control and SEC16A knockdown adipocytes in the basal and 1 nM insulin-stimulated conditions. Endogenous synatxin-6 (Cy3) revealed by IF. Bars, 5 µm. Asterisks indicate nuclei. A.U., arbitrary units; KD, knockdown; WT, wild type. *, P < 0.05, compared to WT unless otherwise indicated.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: Translocation Assay, Transfection, Expressing, Fluorescence, Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: SEC16A KD does not affect insulin signal transduction or TR trafficking. (A) Western blot and corresponding densitometry quantification of AKT and AS160 phosphorylation in response to 1 nM of insulin treatment in control and SEC16A knockdown adipocytes. (B) Quantification of PM to total transferrin receptor (TR) in control and in SEC16A knockdown adipocytes. Data are normalized to the TR surface-to-total value for control adipocytes with 1 nM insulin treatment for each experiment. 40 cells were analyzed per assay. Mean normalized values ± SEM. (C) SEC16A knockdown cells are binned into quartiles based on the integrated residual SEC16A expression (x axis). The mean TR surface-to-total of cells in each quartile, normalized to the control population mean TR surface-to-total (y axis). 120 cells were analyzed. Data are from a representative experiment. Means ± SEM. In all panels, n = 3–5 assays; *, P < 0.05, compared with control unless otherwise indicated. A.U., arbitrary units; KD, knockdown; NS, not significant; WT, wild type.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: Transduction, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: SEC16A independently regulates ER-to-Golgi and GLUT4 trafficking. (A) Quantification of surface to total HA-GLUT4-GFP in control and SEC13, SEC23A, SEC23B, SEC31, or LRRK2 knockdown adipocytes. Data normalized to the HA-GLUT4-GFP surface-to-total value for control adipocytes with 1nM insulin treatment for each experiment. (B) Quantification of PM to total TR distribution in control and SEC23A, SEC23B, or SEC31 knockdown adipocytes. Data normalized to the basal TR surface-to-total value for WT adipocytes. (C) Quantification of relative adipsin secretion at indicated time points after Brefeldin A treatment in control and SEC16A, SEC23A, SEC13, or RAB10 knockdown adipocytes. Data for individual experiments have been normalized to the wild-type 180-min value. Mean normalized values from three to four assays ± SEM, with a linear fit applied, are displayed. (D) Mean rate of adipsin secretion calculated from a linear fit of the data in . Mean normalized values ± SEM, n = 3–4 assays; *, P < 0.05, compared with WT. A.U., arbitrary units; KD, knockdown; WT, wild type.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques:

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: SEC16A encircles perinuclear GLUT4 in adipocytes. (A and B) Representative Airyscan confocal projections of endogenous GLUT4 (green) and endogenous SEC16A (red) in basal (A) and 1 nM of insulin-stimulated adipocytes (B). Endogenous GLUT4 and SEC16A are revealed by IF. Boxed regions magnified in insets illustrates lack of colocalization between GLUT4 and SEC16A in cytosolic puncta. (C and D) Single-plane images, taken from the middle of collected z-stacks, focused on perinuclear region of cells shown in A and B. (E and F) Line-scan quantifications for the indicated lines drawn across the perinuclear region in C and D illustrates that SEC16A tubules are adjacent to perinuclear GLUT4 compartments in basal (E) and insulin-stimulated adipocytes (F). (G and H) Representative Airyscan confocal single plane images of endogenous GLUT4 (Cy3) and transiently expressed SEC16A-GFP in basal (G) and 1 nM of insulin-stimulated adipocytes (H). Endogenous GLUT4 is revealed by IF. Bars, 5 µm. A.U., arbitrary units.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques:

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: The ER marker SEC61B-GFP does not colocalize with perinuclear SEC16A or surround perinuclear GLUT4. (A and B) Representative Airyscan confocal single plane images of basal (A) and insulin-stimulated adipocytes (B) transiently expressing SEC61B-GFP. Endogenous GLUT4 (Cy5) and SEC16A (Cy3) revealed by IF. Boxed regions magnified in insets illustrate individual staining for SEC61B, GLUT4, and SEC16A. Solid arrowheads denote tubules labeled with SEC61B-GFP that are not positive for SEC16A, and arrows denote tubules labeled with SEC16A that are not positive for SEC61B-GFP. Bars, 5 µm.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: Marker, Expressing, Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: SEC16A is not required for maintenance of reticular ER morphology. Representative Airyscan confocal single plane images of SEC61B-GFP transiently expressed in basal and insulin-stimulated control adipocytes (A and B) and SEC16A knockdown adipocytes (C and D). Endogenous SEC16A revealed by indirect immunofluorescence, depicted in the inset. Bars, 5 µm. Asterisks indicate nuclei. KD, knockdown; WT, wild type.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: SEC16A is a RAB10 effector. (A) Representative images of in situ PLA assay performed in SEC16A-GFP and FLAG-RAB10 coexpressing adipocytes in basal and 1 nM insulin-stimulated treatment. Arrows denote SEC16A-GFP and corresponding PLA signal in the perinuclear region. Bars, 5 µm. (B) Quantification of in situ PLA signal (whole cell) shown in A. Data normalized to PLA signal in basal treatment. Mean normalized values ± SEM. (C) Quantification of in situ PLA signal from perinuclear region of cells, shown in A. Mean normalized background subtracted values ± SEM, P = 0.06 (one-tailed Student’s t test). (D) Representative GFP and in situ PLA signal images of cells cotransfected with pEGFP empty vector and FLAG-RAB10 demonstrate negative controls used in experiments. Bar, 10 µm. (E) Quantification of PM to total HA-GLUT4-GFP in adipocytes in which AS160 is stably knocked down and in SEC16A or RAB10 knockdown adipocytes. (F) Quantification of PM to total HA-GLUT4-GFP in control and adipocytes in which RAB10 and SEC16A are knocked down either individually or together. (G) HA-GLUT4-GFP exocytosis in control and RAB10 or SEC16A knockdown adipocytes. Data are cell-associated anti-HA as a function of incubation time at 37°C in medium containing anti-HA antibody. Data are normalized to the 60-min time point for each condition. Mean normalized values ± SEM are shown for a representative experiment. (H) Mean exocytic rate constants determined from assays illustrated in G. Mean normalized values ± SEM, n = 3–6 assays; *, P < 0.05, compared with control. A.U., arbitrary units; eGFP, enhanced GFP; KD, knockdown; NS, not significant; WT, wild type.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: In Situ, One-tailed Test, Plasmid Preparation, Stable Transfection, Incubation

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: SEC16A does not function at the PM to regulate insulin-stimulated GLUT4 translocation. (A) Representative TIRF microscopy images of HA-GLUT4-GFP and endogenous SEC16A (Cy3) in basal and 1 nM insulin-stimulated adipocytes. Bar, 10 µm. (B) Quantification of the colocalization of HA-GLUT4-GFP and endogenous SEC16A in TIRF microscopy. Pearson’s correlation coefficient ( r ) is plotted for the basal and insulin-stimulated conditions. 10 cells were analyzed per condition. (C) Quantification of the integrated intensity of anti-SEC16A (Cy3) fluorescence in TIRF microscopy, normalized to the total epifluorescence intensity of anti-SEC16A (Cy3) in the cell, in basal and insulin-stimulated adipocytes (10 cells per condition). (D) Quantification of the number of SEC16A-positive puncta in TIRF microscopy in the same cells quantified in . Binary masks of SEC16A TIRF puncta above a fixed threshold were generated, and puncta with an area >2 × 2 pixels were counted (10 cells per condition). n = 2–3 assays. A.U., arbitrary units. Mean normalized value ± SEM.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: Translocation Assay, Microscopy, Fluorescence, Generated

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: SEC16A and RAB10 are required for GLUT4 vesicle mobilization from a perinuclear compartment. (A) Representative epifluorescence images of the transferrin washout assay in adipocytes expressing IRAP-TR. Cy3-transferrin (Cy3-Tf) internalized from the medium by IRAP-TR accumulates in the perinuclear region and is released from cells upon incubation in medium without Cy3-Tf. In the top panel, detection of IRAP-TR by immunofluorescence and the corresponding Cy3-Tf images are shown below. Time is in minutes of incubation in medium without Cy3-Tf. Bars, 5 µm. (B) Quantification of Cy3-Tf washout in control adipocytes and adipocytes in which SEC16A or RAB10 were depleted by transient knockdown. Data are normalized to the perinuclear Tf/perinuclear IRAP-TR at time 0 for each experiment. n = 6–8 assays. Two-way analysis of variance: *, P < 0.05 compared with control. (C) Representative Western blots of immunoabsorbed HA-GLUT4-GFP compartments of basal (B) and insulin (I) stimulated adipocytes using 1 or 0.1 μl anti-GFP beads. Proteins coimmunoabsorbed with GLUT4 compartments are in the elution fraction, and proteins not coimmunoabsorbed are in the flow-through fraction. (D) Densitometry quantification of Western blots of immunoabsorbed HA-GLUT4-GFP compartments of basal (B) adipocytes shown in C. Data are coimmunoabsorption of each protein with GLUT4 compartments (band intensity in the eluted fraction) when 1 or 0.1 µl anti-GFP beads is used. For each quantification the data are normalized to the band intensity of coimmunoabsorbed protein using 1 µl beads. n = 3 assays. Mean normalized values ± SEM. (E) Densitometry quantification of Western blots of immunoabsorbed HA-GLUT4-GFP compartments of insulin-stimulated (I) adipocytes shown in C. Data are coimmunoabsorption of each protein with GLUT4 compartments using 1 or 0.1 µl anti-GFP bead. n = 3 assays. Mean normalized values ± SEM. (F) Quantification by Western blot of endogenous RAB10, AS160, TR, and IRAP coimmunoabsorbed with HA-GLUT4-GFP compartments from control and SEC16A depleted adipocytes. Mean normalized values ± SEM; n = 2–5 assays. KD, knockdown; WT, wild type.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: Expressing, Incubation, Immunofluorescence, Western Blot

Journal: The Journal of Cell Biology

Article Title: SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

doi: 10.1083/jcb.201509052

Figure Lengend Snippet: Schematic of the role of RAB10-SEC16A in insulin-regulated GLUT4 translocation. (A) RAB10-SEC16A enhancement of GSV formation generates a constant pool of GSVs of sufficient size to maintain elevated PM GLUT4 in the presence of insulin. (B) Knockdown of RAB10 or its effector SEC16A limits the supply of GSVs, thereby blunting the insulin response. Insulin regulation of other steps of GLUT4 trafficking independent of RAB10 would support a blunted increase of GLUT4 in the PM of RAB10 or SEC16A knockdown cells.

Article Snippet: For experiments assessing colocalization of endogenous SEC16A and SEC16A-GFP with endogenous GLUT4, cells were electroporated with SEC16A-GFP DNA as described, followed by permeabilization with 0.25 mg/ml saponin (Sigma-Aldrich) and indirect immunofluorescence with mouse anti-GLUT4 antibody (2213S; Cell Signaling Technology) or rabbit anti-SEC16A antibody (MKA0310; Cosmo Bio Co.) that was visualized with fluorescently tagged secondary antibody.

Techniques: Translocation Assay