Journal: Cellular oncology (Dordrecht)

Article Title: Single-cell RNA profiling identifies diverse cellular responses to EWSR1/FLI1 downregulation in Ewing sarcoma cells

doi: 10.1007/s13402-021-00640-x

Figure Lengend Snippet: A, Subpopulation studies reveal down-regulation of FAM134B and up-regulation of LC-3-like modifiers (MAPLC3B, GABARAPL2) and ER stress response proteins in cluster C2 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA, error bars indicate S.D.); B, Fluorescence images of EW-8 cells transfected with si-Control or si-EWSR1/FLI1. EW-8 cells were fixed in D1, immunostained and subsequently imaged via fluorescent microscopy to study the induction of the autophagy marker MAPLC3B and the lysosomal marker CD63; C, Quantification of FAM134B co-localization with LCB3, presented as Pearson’s correlation coefficient (r); **** p < 0.0001, t-test, n = 15 fields (~150 cells) in the dormant cells shown in A using the colocalization tool in imageJ; D, EW-8 cells were treated with si-EWSR1/FLI1 or si-Control 48 h before being stained for the ER resident protein SEC61B. The expansion of ER is shown following EWSR1/FLI1 downregulation (boxed areas). E, Quantification of cells with expanded ER after masking nuclei. **** p < 0.0001, t-test, error bars indicate S.D., n = 150 cells.

Article Snippet: As secondary antibody Alexa Fluor 488 (ab150077; Abcam) against FAM134B, SEC61B (sc-393633; Santa Cruz) was used.

Techniques: Fluorescence, Transfection, Control, Microscopy, Marker, Staining

Journal: Nature structural & molecular biology

Article Title: EMC rectifies the topology of multipass membrane proteins.

doi: 10.1038/s41594-023-01120-6

Figure Lengend Snippet: Fig. 4 | Determinants of substrate C-tail translocation. a, 35S-methionine-labeled 23L-SQS variants with the different C-tail lengths indicated (top) translated in the presence of SPCs derived from WT or ΔE cells. Where indicated, 2 µM the Sec61 inhibitor ApraA was included in the translation reaction. Translation reactions were analyzed directly, and substrates with zero, one or two glycans attached are indicated. The two-glycan product is indicative of successful C-tail translocation. C-tail translocation was quantified by calculating the percentage of all glycosylated

Article Snippet: The following antibodies and dilutions were used for blotting: CCDC47 (Bethyl Laboratories, A305-100A; 1:5,000); EMC3 (Invitrogen, 711771; 1:5,000); EMC6 (Abcam, ab84902; 1:1,000); Calnexin (Enzo, ADI-SPA-865; 1:5,000); Sec61α (ref. 70; 1:5,000); TMCO1 (Invitrogen, PA5-43350; 1:500); Sec61β (ref. 71; 1:5,000); CAML (Cell Signaling Technology, 13913S; 1:1,000); FLAG M2-HRP (Sigma, A8592; 1:5,000); EMC4 (Abcam, ab123719; 1:2,000); and β-Actin-HRP (Sigma, A3854; 1:10,000).

Techniques: Translocation Assay, Labeling, Derivative Assay, Glycoproteomics

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) HeLa Fcγ cells were infected with L.p. for the indicated times, stained with anti-Rtn4 (sER marker), Sec61β (rER marker) antibodies, and Hoechst ( L.p .) for immunofluorescence analysis. Results represent three independent experiments (100 vacuoles were scored in each). Error bars represent mean ± SD. Bar, 5 μm. (B) HeLa Fcγ cells stably expressing GFP-Rtn4 and RFP-Sec61β were infected with Halo-tagged WT L.p . for 3 h for FLIP. The top panel represents GFP-Rtn4 before photobleaching, and the bottom represents GFP-Rtn4 after photobleaching for 80 s. Bar, 10 μm. (C) The graph represents the relative fluorescence intensity of GFP-Rtn4 (green lines) and RFP-Sec61β (red lines) in the ER and around LCV. Error bars represent mean ± SD. The purple and red boxes represent the area around LCV and bleach areas, respectively. (D) HeLa Fcγ cells stably expressing GFP-Rtn4 and RFP-Sec61β were infected with WT L.p . for 6 h. The top panel represents RFP-Sec61β before photobleaching, and the bottom represents RFP-Sec61β after photobleaching. The purple and red boxes represent the area around the LCV and bleach areas, respectively. Bar, 10 μm. (E) The graph represents the relative fluorescence intensity of GFP-Rtn4 (green lines) and RFP-Sec61β (red lines) in the ER and around LCV. (F) HeLa Fcγ cells stably expressing GFP-Rtn4 and RFP-Sec61β were infected for 6 h, and RFP-Sec61β was photobleached for FRAP. The green box represents the total area of bleach. The red and blue boxes represent the bleached area around the LCV and the ER, respectively. White arrows represent the area around LCV. Bar, 10 μm. (G) The graph represents the relative fluorescence intensity of GFP-Rtn4 (green lines) and RFP-Sec61β (red lines) in the ER and around LCV. Error bars represent mean ± SD.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Infection, Staining, Marker, Immunofluorescence, Stable Transfection, Expressing, Fluorescence

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) HeLa Fcγ cells were subjected to subcellular fractionation, and immunoblot analysis was done against the indicated proteins. (B) Sucrose gradient fractionation was done to obtain the sER and rER fractions and immunoblotted against indicated antibodies. (C) HeLa Fcγ cells expressing mRFP-Rab4 or mRFP-Rab10 were infected with L.p. for the indicated times, stained with L.p . for immunofluorescence analysis, and the vacuoles positive for mRFP-Rab4 or -Rab10 were quantified. Bar, 5 μm. The purple box represents the magnified image. Results represent three independent experiments (100 vacuoles were scored in each). Error bars represent mean ± SD. (D) HEK293 Fcγ cells were transfected with either mock, Rab4, or Rab10 siRNA for 72 h, and cell extracts were immunoblotted against the indicated antibodies. (E and F) HeLa Fcγ cells were silenced for Rab4 or Rab10 for 72 h, infected with L.p. for 4 and 8 h, and the vacuoles positive for Rtn4 or Sec61β were counted. Results represent three independent experiments, and statistical significance was measured using one-way ANOVA (100 vacuoles were scored in each experiment). Bars represent mean ± SD. (G) HeLa Fcγ cells were silenced with Rab4 and Rab10 siRNA, and the ER morphology was observed by immunofluorescence analysis. Bars, 5 μm. Arrows indicate disruption of the sER tubular network. Arrowheads indicate diffusion of rER to the cell periphery. (H) Triplicate experiments (30 cells for each condition) from 3G were quantitated and are shown as a histogram. Bars represent mean ± SD.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Fractionation, Western Blot, Expressing, Infection, Staining, Immunofluorescence, Transfection, Disruption, Diffusion-based Assay

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) HeLa Fcγ cells were infected with L.p . for the indicated time, fixed, and stained with anti-BAP31 antibody and Hoechst 33342. White boxes indicate the LCV. (B) HeLa Fcγ cells were transfected with either mock or BAP31 siRNA for 72 h and immunoblotted against the indicated antibodies. (C and D) Mock-treated or BAP31-silenced HeLa Fcγ cells were infected with L.p . for the indicated time, fixed, and stained with anti-Rtn4 or -Sec61β antibodies and DAPI, and the vacuoles were quantified. Results are representative of three independent experiments (100 vacuoles were scored in each experiment). Bars represent mean ± SD. (E) HeLa Fcγ cells were silenced with either mock or BAP31 for 72 h, followed by infection with L.p. for 8 h. Post infection, cells were fixed and stained with anti-Rtn4 antibody and Hoechst for immunofluorescence analysis. Bar, 5 μm. (F) HeLa Fcγ cells were silenced with either mock or BAP31 for 72 h, followed by infection with L.p. for 12 h at an MOI of 2. Post infection, cells were fixed and stained with anti- Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. Bar, 5 μm. The graph shows the percentage of vacuoles containing a single bacterium, 1–10 bacteria, 11–30 bacteria, and ≥31 bacteria in a single vacuole. Data are representative of three independent experiments (30 vacuoles were scored in each experiment). Results are shown as the mean ± SD. *** p < 0.001.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Infection, Staining, Transfection, Immunofluorescence, Bacteria

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) Schematic representation of the chromosomal organization of genomic islands of L.p . (B) HeLa Fcγ cells were infected with the indicated genomic island deletion mutant L.p. strains for 4 h and processed for immunofluorescence against BAP31 antibody. The histogram represents the percentage of vacuoles positive for BAP31 post infection with different L.p. strains. (C) HeLa Fcγ cells were transfected with 3xFLAG-Lpg1152 plasmid and processed for immunofluorescence microscopy using FLAG (green) and BAP31 (red) antibodies. Nuclei were stained with DAPI. Bar, 10μm. (D and E) HEK293 Fcγ cells were transfected with the indicated plasmids. Cell extracts were subjected to immunoprecipitation assay post transfection with anti-FLAG antibody and immunoblotted against the indicated antibodies. (F) The docked model complex of Lpg1152 (deep salmon) and BAP31 (green). (G) The electrostatic potential surface of Lpg1152 is shown bound with BAP31 (green). (H) Interacting residues of Lpg1152 (orange, ball-stick) and BAP31 (lime, ball-stick) at the protein-protein interface. (I) HeLa Fcγ cells were infected with the indicated L.p. strains. Histograms represent the infection efficiency, i.e., the percentage of infected cells at MOI 10 1.5 h after infection ( n = 200 per triplicate experiment). (J) HeLa Fcγ cells were infected with the indicated strains of L.p. for the indicated time points. Following infection, LCV positive for BAP31 was scored using immunofluorescence analysis. (K) HeLa Fcγ cells were infected with the indicated strain, and the Rtn4- and Sec61β-positive vacuoles were scored using immunofluorescence. Graphs represent the percentage recruitment of Rtn4 and Sec61β at the indicated time points post infection. (L) HeLa Fcγ cells were infected with the indicated L.p. strains, and the number of L.p. per LCV was counted 8 h after infection ( n = 30 per triplicate experiment). (M) U937 cells were infected with the indicated L.p. strains for 1 h, and colony-forming units (CFU) were counted at the indicated time after infection. Data represent three independent experiments, and statistical significance was carried out with a Student’s t test. Bar represents mean ± SEM.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Infection, Mutagenesis, Immunofluorescence, Transfection, Plasmid Preparation, Microscopy, Staining, Immunoprecipitation

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet: (A) HeLa Fcγ cells were silenced with mock, BAP31, or BAP29 siRNA for 72 h. Post silencing, cells were fixed and stained with anti-Rtn4 and Sec61β antibodies and were subjected to immunofluorescence imaging. Bar, 5 μm. White arrows indicate Sec61β localization to the cell periphery. (B) HeLa Fcγ cells were silenced with either mock or Stx-18 siRNA for 72 h. Post silencing, cells were fixed and stained with anti-Rtn4 and anti-Sec61β antibodies. Bar, 5 μm. (C and D) HeLa Fcγ cells were silenced for Stx18 alone or a combination of Stx18/BAP31 or Stx18/BAP29 siRNA. Following single or double silencing, cell extracts were prepared and immunoblotted against the indicated antibodies. (E) HeLa Fcγ cells were silenced for Stx18 alone, Stx18/BAP31, or Stx18/BAP29; following single or double silencing, cells were fixed and stained with an anti-Rtn4 antibody for immunofluorescence analysis. Bar, 5 μm.

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Staining, Immunofluorescence, Imaging

Journal: Cell reports

Article Title: Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

doi: 10.1016/j.celrep.2024.115053

Figure Lengend Snippet:

Article Snippet: Anti-Sec61β rabbit polyclonal antibody , Proteintech , Cat# 14846-1-AP.

Techniques: Virus, Recombinant, Transfection, In Situ, Fluorescence, Stable Transfection, Expressing, Software

Journal: bioRxiv

Article Title: Meta-organization of Translation Centers Revealed by Proximity Mapping of Endoplasmic Reticulum Ribosome Interactors

doi: 10.1101/398669

Figure Lengend Snippet: Cytoscape plots of enriched proteins for the Sec61β, LRRC59 and Ribophorin I BioID reporter cell lines reveal different functional enrichments for proximally labeled proteins. Center nodes indicate the chimera protein from each cell line while the surrounding nodes develop from the proteomic datasets. Sizes indicate ranked normalized counts with the largest nodes having the highest values. Green nodes indicate stable, well-characterized protein oligomeric complexes, dark blue nodes are proteins comprising the top GO category indicated underneath each plot and in the appended table. Asterisks denote proteins that are in both established oligomeric complexes and GO categories. Borders indicate whether the protein is a membrane or soluble protein.

Article Snippet: Primaries used: BirA (Abcam #14002, polyclonal, chicken IgG), TRAPα ( )(polyclonal, rabbit IgG), tubulin (Iowa Hybridoma Bank, E7, monoclonal, mouse IgG, Iowa City, IA), Sec61β (Gift of Ramanujan Hegde, University of Cambridge, polyclonal, rabbit IgG), LRRC59 (Bethyl Labs A305-076A, polyclonal, rabbit IgG, Montgomery, TX), Sec62 (gift from Richard Zimmerman, polyclonal, rabbit IgG), Ribophorin I ( )(polyclonal, rabbit IgG), streptavidin-RD680 (Li-Cor, P/N 925-68079).

Techniques: Functional Assay, Labeling, Membrane

Journal: bioRxiv

Article Title: Meta-organization of Translation Centers Revealed by Proximity Mapping of Endoplasmic Reticulum Ribosome Interactors

doi: 10.1101/398669

Figure Lengend Snippet: A) The three chimera proteins, Ribophorin I (RPN1), LRRC59 and SEC61β (SEC61B), implicated in ribosome binding, with common, shared proteins. B) Cytoscape plot of shared proteins confirms several established protein-protein interactions from experimental evidence (dotted edge lines, bold lines if connected to a reporter node). Size of the nodes are based on highest normalized count from the shared reporters. Proteins shared by specific chimera are distinguished by the indicated color scheme. LRRC59 share more proteins with the SEC61B and RPN1 reporters than SEC62. Files containing all membrane protein raw MS data is contained in Scaffold files as – source data 1 and 2.

Article Snippet: Primaries used: BirA (Abcam #14002, polyclonal, chicken IgG), TRAPα ( )(polyclonal, rabbit IgG), tubulin (Iowa Hybridoma Bank, E7, monoclonal, mouse IgG, Iowa City, IA), Sec61β (Gift of Ramanujan Hegde, University of Cambridge, polyclonal, rabbit IgG), LRRC59 (Bethyl Labs A305-076A, polyclonal, rabbit IgG, Montgomery, TX), Sec62 (gift from Richard Zimmerman, polyclonal, rabbit IgG), Ribophorin I ( )(polyclonal, rabbit IgG), streptavidin-RD680 (Li-Cor, P/N 925-68079).

Techniques: Binding Assay, Protein-Protein interactions, Membrane

Journal: bioRxiv

Article Title: Meta-organization of Translation Centers Revealed by Proximity Mapping of Endoplasmic Reticulum Ribosome Interactors

doi: 10.1101/398669

Figure Lengend Snippet: A) Experimental schematic illustrating the biotin-tagged ribosome isolation and analysis protocol. B) Velocity sedimentation analysis of ribosomal small and large subunits derived from LRRC59 and Sec61β BioID reporter cell lines. Illustrated are the A 254 traces, RNA gel micrographs depicting 18S and 28S rRNAs. Also illustrated are streptavidin blots of the small and large ribosomal subunits from the reporter cell lines, with 80S ribosomes as comparison. C) To determine if BioID-mediated biotinylation altered ribosome function, polyribosomes were fractionated by sucrose gradient velocity sedimentation and biotin-labeled protein distributions analyzed by streptavidin blots of the precipitated gradient fractions. RNA gels of the gradient fractions are included to confirm ribosome migration.

Article Snippet: Primaries used: BirA (Abcam #14002, polyclonal, chicken IgG), TRAPα ( )(polyclonal, rabbit IgG), tubulin (Iowa Hybridoma Bank, E7, monoclonal, mouse IgG, Iowa City, IA), Sec61β (Gift of Ramanujan Hegde, University of Cambridge, polyclonal, rabbit IgG), LRRC59 (Bethyl Labs A305-076A, polyclonal, rabbit IgG, Montgomery, TX), Sec62 (gift from Richard Zimmerman, polyclonal, rabbit IgG), Ribophorin I ( )(polyclonal, rabbit IgG), streptavidin-RD680 (Li-Cor, P/N 925-68079).

Techniques: Isolation, Sedimentation, Derivative Assay, Comparison, Labeling, Migration

Journal: bioRxiv

Article Title: Meta-organization of Translation Centers Revealed by Proximity Mapping of Endoplasmic Reticulum Ribosome Interactors

doi: 10.1101/398669

Figure Lengend Snippet: A) Schematic of experimental protocol for capturing biotin-labeled polysomes. B) Final subcellular distributions of proteins encoded by ribosome-associated RNAs. Stack plots of RNA-seq TPM reveal an enrichment for organellar membrane proteins (DeepLoc1.0) compared to the total mRNA distribution by TPM for membrane and total cell (LocTree3, using datasets from . The transcriptomes from Sec61β and LRRC59 labeled ribosomes diverge from the total and secretory/membrane distributions, and from one another. C) TPM analysis of the enriched and shared mRNAs for the Sec61β datasets showing both subcellular distributions and membrane prediction of the encoded proteins. TPM analysis of the enriched and shared mRNAs for the LRRC59 datasets showing both subcellular distributions and membrane prediction of the encoded proteins. Plasma membrane (PM); Transcripts per million (TPM). E) Read count analysis of raw counts shown for each of the datasets by percentage of those that aligned to the human genome and counted by htseq-count as described in the methods. F) Table of top ten genes by fold change value for enriched and shared datasets color coded by fold enrichment over the control datasets.

Article Snippet: Primaries used: BirA (Abcam #14002, polyclonal, chicken IgG), TRAPα ( )(polyclonal, rabbit IgG), tubulin (Iowa Hybridoma Bank, E7, monoclonal, mouse IgG, Iowa City, IA), Sec61β (Gift of Ramanujan Hegde, University of Cambridge, polyclonal, rabbit IgG), LRRC59 (Bethyl Labs A305-076A, polyclonal, rabbit IgG, Montgomery, TX), Sec62 (gift from Richard Zimmerman, polyclonal, rabbit IgG), Ribophorin I ( )(polyclonal, rabbit IgG), streptavidin-RD680 (Li-Cor, P/N 925-68079).

Techniques: Labeling, RNA Sequencing, Membrane, Clinical Proteomics, Control

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: Nuclear β-DG derives from the PM. ( A ) C2C12 cells were incubated with biotin for the indicated time intervals to label cell surface proteins (see Methods). Cells were then subjected to subcellular fractionation to isolate nuclear and non-nuclear fractions and biotinylated proteins were pulled-down using streptavidin-agarose beads and analyzed by SDS-PAGE/Western blotting using primary antibodies for non-phosphorylated-β-DG (upper panel) and phosphorylated β-DG (lower panel). Lower panel blot was reorganized so that the time points order matched the ones shown in upper panel, original blot is shown in Supp. Figure . Input: immunoblotting analysis of cellular fractions prior to streptavidin-mediated pull-down. B: Bound/precipitated fraction. Membranes were stripped and reprobed for lamin A/C and GAPDH/calnexin as purity controls for nuclear and non-nuclear fractions respectively. ( B ) C2C12 cells cultured on glass coverslips were double-immunostained for total β-DG and the early endosomal marker EEA1. Nuclei were counterstained with DAPI prior to CLSM analysis. A typical single Z-section from three independent experiments is shown. Scale bar 20 µm.

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Incubation, Fractionation, SDS Page, Western Blot, Cell Culture, Marker

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: Retrograde trafficking of β-DG from the PM to the ER. ( A ) ER was purified using density gradient techniques (OptiPrep) and then ER fractions were immunoblotted for the ER marker calnexin or β-DG on the same membrane. ( B ) Verification of the purity of ER fractions: Aliquots from each step of the ER purification were analyzed by Western blotting using primary antibodies against EEA1 (early endosomal marker), GAPDH (cytosolic marker) and Sp3 (nuclear marker). As a PM marker, ER was isolated from biotinylated cells at 4 °C, the lysates were pulldown using streptavidin-agarose beads and then blotted with HRP-streptavidin. NF: Nuclear fraction; NN: Non-nuclear fraction; CS: Cytosolic fraction; ER: Endoplasmic reticulum fraction. ( C ) Cells were subjected to cell surface biotinylation and subsequently to ER purification using the OptiPrep gradient. The ER fractions were combined and biotinylated proteins were precipitated using streptavidin-agarose beads and then analyzed by SDS-PAGE/Western blotting with antibodies against β-DG and calnexin.

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Purification, Marker, Membrane, Western Blot, Isolation, SDS Page

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: Retrograde trafficking of β-DG from the PM to the ER. ( A ) ER was purified using density gradient techniques (OptiPrep) and then ER fractions were immunoblotted for the ER marker calnexin or β-DG on the same membrane. ( B ) Verification of the purity of ER fractions: Aliquots from each step of the ER purification were analyzed by Western blotting using primary antibodies against EEA1 (early endosomal marker), GAPDH (cytosolic marker) and Sp3 (nuclear marker). As a PM marker, ER was isolated from biotinylated cells at 4 °C, the lysates were pulldown using streptavidin-agarose beads and then blotted with HRP-streptavidin. NF: Nuclear fraction; NN: Non-nuclear fraction; CS: Cytosolic fraction; ER: Endoplasmic reticulum fraction. ( C ) Cells were subjected to cell surface biotinylation and subsequently to ER purification using the OptiPrep gradient. The ER fractions were combined and biotinylated proteins were precipitated using streptavidin-agarose beads and then analyzed by SDS-PAGE/Western blotting with antibodies against β-DG and calnexin.

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Purification, Marker, Membrane, Western Blot, Isolation, SDS Page

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: Src-dependent Tyrosine 890 phosphorylation of β-DG drives its nuclear targeting. ( A ) C2C12 cells cultured on glass coverslips were treated with sodium orthovanadate (OV) for 3 h, fixed and immunostained using a phospho-specific antibody that recognizes Tyr 890 phosphorylated β-DG. Nuclei were stained with DAPI (blue color) prior to CLMS analysis, with single optical Z-sections. Scale bar = 20 μm. ( B ) Cultures from OV-treated cells were fractionated into total (T), cytoplasmic ( C ) and nuclear (N) extracts and these extracts were separated by SDS-PAGE and subjected to Western blot analysis using phospho-β-DG antibodies. Nuclear (lamin B1) and cytoplasmic (GAPDH) protein markers were analyzed in parallel to validate the purity of the fractions. ( C ) C2C12 cells grown on glass coverslips were treated with PP2 or vehicle, fixed and immunolabeled for total β-DG and counterstaining with DAPI to visualize nuclei. Cells were imaged by CLMS and typical single Z-sections are shown (scale bar is 20 µm). Right panel. Quantitative analysis of CLMS images was carried out to obtain the nuclear to cytoplasmic fluorescence intensity of total β-DG (F n/c, see Methods). Results represent mean +/− SD for 3 separate experiments, with significant differences denoted by the p value (Student t-test). ( D ) Nuclear and cytoplasmic fractions obtained from control and PP2-treated cells were analyzed by SDS-PAGE/Western blotting using specific antibodies against total β-DG. Sp3 and GAPDH were used as loading controls for nuclear and cytoplasmic fractions respectively. Right panel. Nuclear to cytoplasmic ratio (n/c) of total β-DG was measured by densitometry analysis and data correspond to mean +/− SD for 3 independent experiments, with significant differences indicated by the p value (Student t-test). ( E ) C2C12 cells grown on glass coverslips were transiently transfected to express DGWT-GFP, DGY890E-GFP or DGY890F-GFP proteins. At 24 h post-transfection, cells were fixed, stained with DAPI and subjected to CLMS, with typical single optical Z-sections shown. Scale bar is 20 µM. Right panel. The F n/c ratio for DGWT-GFP, DGY890E-GFP and DGY890F-GFP was obtained and plotted. Results correspond to mean +/− SD for 3 separate experiments (n = 50 cells), with significant differences indicated by the p value (Student t-test).

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Phospho-proteomics, Cell Culture, Staining, SDS Page, Western Blot, Immunolabeling, Fluorescence, Control, Transfection

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: β-DG traffics from ER to Golgi prior to its nuclear localization. ( A ) C2C12 cells cultured on glass coverslips were transiently transfected to express the galactosyl-transferase-CFP fusion protein (Golgi-CFP) and 16 h post-transfection they were treated with BFA or vehicle alone (DMSO; see Methods). Afterwards, cells were fixed, stained with DAPI and further analyzed by CLSM (upper panel). C2C12 cells seeded on coverslips were treated with vehicle or BFA, fixed, immunolabeled for non-phosphorylated β-DG and counterstained with DAPI for nuclei visualization prior to analysis by CLSM (lower panel). Maximum intensity projections are shown. The fluorescence intensity in the nucleus and cytoplasm (F n/c ratio) was quantified to estimate β-DG nuclear accumulation (see Methods). Data shown in the graph (right) represent the mean +/− SD from three separate experiments (n = 30 cells), with p value denoting statistical significance (Student t-test). Scale bar 20 µm. ( B ) Control and BFA-treated cells were fractionated into cytoplasmic and nuclear extracts and further subjected to SDS-PAGE/Western blotting analysis using anti-β-DG antibodies for the immunodetetection of total β-DG. Membranes were striped and reprobed for calnexin (cytoplasmic marker) and lamin B1 (nuclear marker). Densitometric analysis of immunoblot autoradiograms was carried out and the relative levels of β-DG in the nucleus and cytoplasmic (n/c) were obtained. Results represent the mean +/− SD of 4 separate experiments, with p values indicating significant differences (Student t-test). ( C ) Vehicle- or BFA-treated cells were fractionated into cytosolic and total membrane extracts prior to being subjected to SDS-PAGE/Western blotting analysis, using anti-β-DG antibodies for total β-DG. Stripped membranes were reprobed for caveolin (membrane protein) and actin (cytosolic marker).

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Cell Culture, Transfection, Staining, Immunolabeling, Fluorescence, Control, SDS Page, Western Blot, Marker, Membrane

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: Nuclear β-DG derives from the PM. ( A ) C2C12 cells were incubated with biotin for the indicated time intervals to label cell surface proteins (see Methods). Cells were then subjected to subcellular fractionation to isolate nuclear and non-nuclear fractions and biotinylated proteins were pulled-down using streptavidin-agarose beads and analyzed by SDS-PAGE/Western blotting using primary antibodies for non-phosphorylated-β-DG (upper panel) and phosphorylated β-DG (lower panel). Lower panel blot was reorganized so that the time points order matched the ones shown in upper panel, original blot is shown in Supp. Figure . Input: immunoblotting analysis of cellular fractions prior to streptavidin-mediated pull-down. B: Bound/precipitated fraction. Membranes were stripped and reprobed for lamin A/C and GAPDH/calnexin as purity controls for nuclear and non-nuclear fractions respectively. ( B ) C2C12 cells cultured on glass coverslips were double-immunostained for total β-DG and the early endosomal marker EEA1. Nuclei were counterstained with DAPI prior to CLSM analysis. A typical single Z-section from three independent experiments is shown. Scale bar 20 µm.

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Incubation, Fractionation, SDS Page, Western Blot, Cell Culture, Marker

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: Inhibition of dynamin-dependent endocytosis reduces nuclear localization of β-DG. ( A , left panel) Serum-starved C2C12 cells, seeded on coverslips, were treated with 40 µM dynasore (endocytosis inhibitor) or 0.05% DMSO (vehicle) for 30 min at 37 °C and then incubated for 5 min at 20 °C with Alexa594-transferrin (red). After 15 min at 37 °C, cells were fixed, stained with DAPI (nuclei) and transferrin uptake was monitored by CLSM analysis, with typical images shown (scale bar is 20 µm). ( A , right panel) Cells treated with dynasore or DMSO, as above, were immunolabeled for β-DG and counterstained with DAPI prior to be imaged by CLSM, with typical Z-sections shown (scale bar is 20 µm). (A, lower panel). Nuclear accumulation of β-DG (F n/c) was estimated as described in Methods. Data correspond to mean +/− SD from a series of three separate experiments (n = 30 cells). ( B ) Cytoplasmic and nuclear extracts obtained from dynasore- or DMSO-treated cells were analyzed by Western blotting using anti-β-DG antibodies. Stripped membranes were reprobed for lamin A/C and GAPDH as purity and loading controls for nuclear and cytoplasmic extracts respectively. The nuclear/cytoplasmic ratio (n/c) of β-DG was estimated by densitometry analysis. Results represent the mean +/− SD for 3 separate experiments, with significant differences denoted by p values (Student t-test). ( C ) DMSO- and dynasore-treated cells were subjected to biotinylation assays as in Fig. . At 1 h post-biotinyation time, cells were fractionated into nuclear and non-nuclear fractions and pulled-down using streptavidin-agarose beads. Recovered and unbound proteins were analyzed by SDS-PAGE/Western blotting using primary antibodies for total β-DG. Membranes were stripped and reprobed for lamin A/C and calnexin; markers for nuclear and non-nuclear fractions respectively. Input: immunoblotting analysis of cellular fractions prior to pull-down. B, bound/precipitated fraction. Un, unbound fraction.

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Inhibition, Incubation, Staining, Immunolabeling, Western Blot, SDS Page

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: Retrograde trafficking of β-DG from the PM to the ER. ( A ) ER was purified using density gradient techniques (OptiPrep) and then ER fractions were immunoblotted for the ER marker calnexin or β-DG on the same membrane. ( B ) Verification of the purity of ER fractions: Aliquots from each step of the ER purification were analyzed by Western blotting using primary antibodies against EEA1 (early endosomal marker), GAPDH (cytosolic marker) and Sp3 (nuclear marker). As a PM marker, ER was isolated from biotinylated cells at 4 °C, the lysates were pulldown using streptavidin-agarose beads and then blotted with HRP-streptavidin. NF: Nuclear fraction; NN: Non-nuclear fraction; CS: Cytosolic fraction; ER: Endoplasmic reticulum fraction. ( C ) Cells were subjected to cell surface biotinylation and subsequently to ER purification using the OptiPrep gradient. The ER fractions were combined and biotinylated proteins were precipitated using streptavidin-agarose beads and then analyzed by SDS-PAGE/Western blotting with antibodies against β-DG and calnexin.

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Purification, Marker, Membrane, Western Blot, Isolation, SDS Page

Journal: Scientific Reports

Article Title: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

doi: 10.1038/s41598-017-09972-x

Figure Lengend Snippet: Nuclear translocation of β-DG is dependent on the Sec61 translocon. ( A ) C2C12 cells were transiently transfected to express Sec61β-GFP or GFP alone. The transfected cells were lysed 8 h post-transfection and immunoprecipitated using the GFP-Trap system; the precipitated proteins were analyzed by Western blot using antibodies against β-DG. Input corresponds to 5% of protein extract prior to immunoprecipitation; Un, unbound proteins; B, bound proteins. ( B ) Lysates from C2C12 cells stably transfected with vectors encoding shRNAs directed against mouse Sec61β mRNA (Sec61β shRNA1 and 2) or a scrambled shRNA (control) were analyzed by western blotting using antibodies against Sec61β and calnexin (loading control). ( C ) C2C12 cells expressing the scrambled shRNA or the Sec61β shRNAs (1 or 2) were cultured on glass coverslips, fixed, immunostained for total β-DG and counterstained with DAPI for nuclei visualization, prior to being analyzed by CLSM, with typical single Z-sections shown (scale bar is 20 µm). Quantitative analysis of the levels of β-DG nuclear accumulation (Fn/c ratio) was performed (right panel) and results represent the mean +/− SD for three separate experiments (n ≥ 50), with significant differences denoted by the p values (Student’s t-test). ( D ) Cells were fractionated to obtain cytosolic and membrane extracts. Distribution of Sec61β was analyzed by SDS-PAGE/Western blotting analysis using anti-Sec61β antibodies. Purity of cell fractions was analyzed by using primary antibodies against caveolin (membrane marker) and actin (cytosolic marker). C, cytosolic fraction; M, membrane fraction.

Article Snippet: Rabbit polyclonal antibodies against: calnexin, lamin A/C, GAPDH, Sp3, lamin B1 and EEA1, a goat polyclonal antibody against caveolin (Santa Cruz Biotechnology (Santa Cruz, CA., USA) and a mouse monoclonal antibody against Sec. 61β (Abcam, Cambridge, UK) were utilized.

Techniques: Translocation Assay, Transfection, Immunoprecipitation, Western Blot, Stable Transfection, shRNA, Control, Expressing, Cell Culture, Membrane, SDS Page, Marker