Journal: The Journal of Neuroscience

Article Title: Fas/Apo [Apoptosis]-1 and Associated Proteins in the Differentiating Cerebral Cortex: Induction of Caspase-Dependent Cell Death and Activation of NF-κB

doi: 10.1523/jneurosci.19-05-01754.1999

Figure Lengend Snippet: Figure 6. The expression of the Fas-associated proteins RIP (A), FADD (B), FLIP (C), and FasL (D) was examined by Western analysis of 50 mg of detergent-soluble protein obtained from P6 cerebral cortex obtained from four separate animals (lanes 1-4). Bands corresponding to RIP (74 kDa) and FasL (37 kDa) were detected in 10 mg of Jurkat cell lysates (a positive control) and in P6 cortex. FLIP expression in P6 cortex was confirmed by the appearance of a similar band just below the 66 kDa molecular weight marker, corresponding to the expected size of ;60 kDa, in protein extract from COS7 cell transfected with the full-length FLIP plasmid (positive control). In contrast, we did not detect any bands corresponding to the expected size for FADD in the cerebral cortex, although a band of the approximate size (24 kDa) was observed in Jurkat cell-derived protein (a positive control). Mkr, Biotinylated marker.

Article Snippet: Binding of the second, primary antibody was visualized by the appropriate secondary antibody, either a biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch, 1:500) or horse anti-mouse (Vector Laboratories, 1:1000) coupled to either streptavidin-FITC or rhodamineavidin.

Techniques: Expressing, Western Blot, Positive Control, Molecular Weight, Marker, Transfection, Plasmid Preparation, Derivative Assay

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: A Biotinylated Affinity-Labeling Agent for RBPs

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: Labeling

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: 2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1, at 10 μM, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: SDS Page, Labeling, Two-Dimensional Gel Electrophoresis, Stripping Membranes, Silver Staining, Incubation, Avidin-Biotin Assay

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1, at 5 μM, for 20 s at 4 °C; lane 4, RPE incubated with 1, at 5 μM, for 2 min at 4 °C; lane 5, RPE incubated with 1, at 5 μM, for 10 min at 4 °C; lane 6, RPE incubated with 1, at 5 μM, for 30 min at 4 °C; lane 7, RPE incubated with 1, at 5 μM, for 1 h at 4 °C; and lane 8, RPE incubated with 1, at 5 μM, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: Labeling, Avidin-Biotin Assay, Transferring, SDS Page, Incubation

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1, at 5 μM, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1, at 5 μM, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1, at 5 μM, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1, at 5 μM, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μM, 1 h) and then 1, at 5 μM, for 10 min at 4 °C; lane 7, RPE labeled with 1, at 5 μM, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: Labeling, Avidin-Biotin Assay, SDS Page, Incubation

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: Elution of biotinylated proteins from neutravidin–agarose beads. The neutravidin–agarose beads which contain the labeled RPE were treated with buffer at either pH 7.5 or 11: lane 1, pH 11 for 6 h; lane 2, pH 7.5 for 6 h; lane 3, pH 11 overnight; and lane 4, pH 7.5 overnight.

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: Labeling

Journal: PLoS ONE

Article Title: Evidence for SH2 Domain-Containing 5′-Inositol Phosphatase-2 (SHIP2) Contributing to a Lymphatic Dysfunction

doi: 10.1371/journal.pone.0112548

Figure Lengend Snippet: ( A )Western blot analysis depicting expression of SHIP2 (green) and DYKDDDDK (FLAG) tag epitope (red) in SHIP2-FLAG TIME transfectants. COX IV (red) used to determine equal protein loading. ( B ) T180A-SHIP2 exhibits reduced migration assessed by wound healing (top panels) and tube formation (bottom panels). ( C ) WT-SHIP2 exhibits increased chemotaxis compared to Vector in response to HGF while T180A-SHIP2 has slightly reduced, but not significant, chemotaxis towards HGF, compared to WT. Data are presented as % of area covered by migrated cells on underside of transwell filters and cells stained with DRAQ5. ( D ) WT-SHIP2 exhibits increased adhesion (assessed by crystal violet staining) over Vector on BSA and collagen while T180A-SHIP2 shows decreased adhesion to collagen compared to WT. ( E ) T180A-SHIP2 results in increased activation of AKT and ERK upon HGF stimulation in comparison to WT-SHIP2. Representative blots shown. Phosphorylation was determined by Western blotting of cell lysates with phosphospecific antibodies. Blots were reprobed with total AKT and ERK antibodies to demonstrate equal loading. Phospho- and total-antibody reprobes were detected with IRDye 680 nm secondary antibodies. Quantification of AKT and ERK activation performed by average mean fluorescence intensity (MFI) of 3 independent experiments and represented as a ratio of pAKT-S473 to total AKT and pERK1/2 to total ERK1/2, respectively. ( F ) WT-SHIP2 results in less VEGFC-induced PIP3 levels compared to Vector, while T180A has increased PIP3 levels compared to WT and data expressed as fluorescence intensity of PIP3 normalized to 1% FBS in 100 cells/experiment (N = 3). PIP3 levels were assessed by fluorescent immunocytochemistry of 10 min VEGFC-stimulated cells using anti-PIP3 antibody. ( G ) Malachite green in vitro phosphatase assay indicates no significant difference in amount of phosphate released by T180A SHIP2 over WT-SHIP2 suggesting similar enzymatic activity against recombinant PIP3 substrate. Recombinant SHIP2 (rSHIP2) and PIP3 (rPIP3 only, no enzyme) used as positive and negative controls respectively. Comparisons are between Vector vs . WT; WT vs . T180A and rSHIP2 vs. WT; and rSHIP2 vs. rPIP3 * p <0.05, ** p <0.01, *** p <0.001. Scale bar = 100 µm ( B ) and 50 µm ( C and D ) MW = molecular weight marker.

Article Snippet: Immunofluorescence cytochemistry was done using rabbit anti-SHIP2 antibody (Cell Signal) for determining expression of SHIP2 in LEC and anti-PIP3 IgG antibody (Echelon Biosciences) to determine cellular levels of PtdIns(3,4,5)P3.

Techniques: Western Blot, Expressing, FLAG-tag, Migration, Chemotaxis Assay, Plasmid Preparation, Staining, Activation Assay, Comparison, Phospho-proteomics, Fluorescence, Immunocytochemistry, In Vitro, Phosphatase Assay, Activity Assay, Recombinant, Molecular Weight, Marker

Journal:

Article Title: v-SNARE-dependent secretion is required for phagocytosis

doi:

Figure Lengend Snippet: Changes in macrophage surface area after phagocytosis of OPZ. (A–C) J774 cells were allowed to interact with biotinylated, Texas red-labeled OPZ. Extracellular particles were identified by treatment with avidin-FITC. (A) Bright field (Nomarski) image. Bar, 10 μm. (B) Red fluorescence (Texas red emission) of cells in A as analyzed by confocal fluorescence microscopy, indicating the location of OPZ. (C) Green fluorescence (avidin-FITC emission) of cells in A, identifying extracellular OPZ. (D–F) J774 cells were allowed to internalize Texas red-labeled OPZ as in A, treated with the dye FM-143 at 4°C (which provides a measure of the surface membrane), and then examined by confocal microscopy. (D) Bright field image; inset shows an extracellular particle (arrow) and an intracellular particle (arrowhead). (E) Red (Texas red) fluorescence of cells in D indicating the location of OPZ. (F) Green (FM-143) fluorescence indicating surface labeling of cell only. (G) Analysis of association of OPZ with J774 cells by flow cytometry. The arbitrarily designated quadrants define the following populations: i, extracellular OPZ; ii, macrophages with associated OPZ; iii, debris; and iv, macrophages not associated with OPZ. (H) Frequency histogram of the fluorescence intensity of cell-associated FM-143, a measure of cell surface area. The lighter line (a) corresponds to cells in quadrant iv in G, which had no associated OPZ. The heavy line (b) corresponds to cells in quadrant ii in G, which had internalized OPZ. (I) Correlation between Texas red fluorescence (abscissa), which is proportional to the number of OPZ particles (fluorescence equivalent to one internalized particle shown by arrow) and the uptake of FM-143 (left ordinate), which is proportional to surface area (calculated from the volume of cells, assuming a smooth spherical surface; right ordinate). Data from five separate experiments.

Article Snippet: Texas red-labeled zymosan, NHS-biotin, avidin-fluorescein isothiocyanate, FM-143, Lucifer yellow, FITC, rhodamine-labeled human holo-transferrin, tetramethylrhodamine B isothiocyanate, and Texas red-dextran were obtained from Molecular Probes.

Techniques: Labeling, Avidin-Biotin Assay, Fluorescence, Microscopy, Confocal Microscopy, Flow Cytometry

Journal:

Article Title: v-SNARE-dependent secretion is required for phagocytosis

doi:

Figure Lengend Snippet: Impaired phagocytosis after degradation of VAMP-2 in J774 cells. (A–C) J774 cells were injected with TeTx-LC in a solution containing fixable FITC-dextran as a marker of injection. After 5 h of incubation at 37°C, the cells were stained with antibody to VAMP-2. (A) Bright field micrograph. Bar, 10 μm. (B) Green fluorescence (Lucifer yellow emission) of the field shown in A identifying two injected cells. (C) VAMP-2 immunostaining in the cells shown in A. (D–F) J774 cells were injected with tetanus toxin and FITC-dextran as above, then assessed for their ability to perform phagocytosis of opsonized SRBCs. (D) Bright field micrograph. Inset (D′) shows cell injected with dextran alone. Arrow indicates location of representative internalized SRBCs. (E and E′) Green fluorescence emission of the cells shown in D. Arrowhead demonstrates the displacement of FITC-dextran by internalized the SRBCs. (F) Effect of toxin injection on phagocytosis. Data are means ± SE of 250 noninjected control (Ctl) cells, 175 buffer- (Buf) injected controls, 244 TeTx-LC- (Tet) injected cells, and 75 botulinum- (Bot) injected cells (asterisks indicate P < 0.05 vs. Ctl). (G–I) J774 cells were injected with TeTx-LC as above and then assessed for their ability to bind opsonized SRBCs. (G) Bright field micrograph. (H) Fluorescence emission of the cells shown in G identifying injected cells. (I) Effect of toxin injection on SRBC adherence to J774 cells. Data are means ± SE of 165 noninjected control (Ctl) cells, 64 buffer- (Buf) injected controls, 75 TeTx-LC- (Tet) injected cells, and 57 botulinum- (Bot) injected cells. Images are representative of five separate experiments.

Article Snippet: Texas red-labeled zymosan, NHS-biotin, avidin-fluorescein isothiocyanate, FM-143, Lucifer yellow, FITC, rhodamine-labeled human holo-transferrin, tetramethylrhodamine B isothiocyanate, and Texas red-dextran were obtained from Molecular Probes.

Techniques: Injection, Marker, Incubation, Staining, Fluorescence, Immunostaining

Journal:

Article Title: v-SNARE-dependent secretion is required for phagocytosis

doi:

Figure Lengend Snippet: Effect of tetanus toxin on endocytosis. (A and B) J774 cells were injected with TeTx-LC and fluorescein-dextran as an injection marker, incubated for 5 h at 37°C, and then allowed to internalize Texas red-dextran for 2 h at 37°C. (A) Typical confocal micrograph demonstrating Texas red fluorescence. (B) Fluorescein-dextran emission identifying injected cell. (C–F) FcγRIIA-expressing CHO cells were transiently transfected with cDNA for TeTx-LC and GFP and allowed to internalize either rhodamine-labeled transferrin (C and D) or tetramethylrhodamine B isothiocyanate-labeled aggregated human IgG (E and F) for 1 h at 37°C. (C and E) Confocal fluorescence micrograph illustrating the distribution of transferrin (C) or aggregated IgG (E). (D and F) GFP emission identifying transfected cells. Representative of three experiments, with at least 50 injected or transfected cells per experiment. Bar, 10 μm.

Article Snippet: Texas red-labeled zymosan, NHS-biotin, avidin-fluorescein isothiocyanate, FM-143, Lucifer yellow, FITC, rhodamine-labeled human holo-transferrin, tetramethylrhodamine B isothiocyanate, and Texas red-dextran were obtained from Molecular Probes.

Techniques: Injection, Marker, Incubation, Fluorescence, Expressing, Transfection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Location of missense and nonsense mutations in SLC9A6/NHE6 variants of patients with Christianson syndrome. A, schematic planar drawing of the predicted membrane topology of the longest splice-variant of mammalian NHE6 and location of the mutations (yellow circles). Two consensus N-linked glycosylation sites (128NVT and 145NVS) within extracellular loop 2 have been verified experimentally (data not shown) and are illustrated in the drawing. B, phylogenetic comparison of the primary sequence of segments containing the various mutations in NHE6. The affected residues are shaded in black.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Variant Assay, Glycoproteomics, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Structure homology modeling of transmembrane NHE6 variants associated with Christianson syndrome. A, front (membrane aspect, left panel) and top (extracellular aspect, right panel) views of a 3D structure homology model of dimeric human NHE6 based on the crystal structure of the bacterial T. thermophilus Na+/H+ antiporter NapA (TtNapA) (Protein Data Bank accession code 5bz3; 2.30 Å, 15% identity, 27% similarity), which provided the broadest coverage, highest resolution, and best spatial fit compared with other crystallized bacterial Na+/H+ antiporters. The proposed structure includes only the membrane-spanning helices (M2–M12; amino acids 74–540) that aligned with homologous segments of TtNapA. The top view includes the locations of the transmembrane-localized residues Leu-188 and Gly-383 mutated in CS. B, molecular dynamics simulation of structural changes predicted to occur in TM4 (highlighted in cyan) upon substitution of Leu-188 with Pro (L188P). The monomeric forms of NHE6 WT and L188P are illustrated. C and D, structural perturbations predicted to occur in the intramembranous re-entrant (R) loop between helices M8 and M9 upon substitution of Gly-383 with Asp (G383D). C, upper and lower panels show front and top views, respectively, of Gly-383, which is packed tightly against amino acids Phe-373, Ala-376, and Glu-377 (top left). Mutation of Gly-383 to Asp would result in steric clashes between these residues and interfere with the packing between these segments of the R-loop. D, G383D substitution would also disrupt interactions of R-loop residues Trp-379, Phe-381, and especially Thr-382 with residues Asp-92 and Ile-296 in helix M7 and residues Ile-330, Phe-331, and Ser-334 in helix M8.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the biosynthetic maturation of NHE6 variants. A, AP-1 cells transiently expressing NHE6GFP WT or CS-linked variants were lysed at the indicated time points (6–48 h) post-transfection. Equal amounts of proteins (20 μg) were subjected to SDS-PAGE and immunoblotting with a polyclonal anti-GFP antibody. NHE6 migrates as multiple bands: higher molecular weight bands represent the fully-glycosylated (fg) and core-glycosylated (cg) dimeric (d) forms of the exchanger that do not fully dissociate under SDS-PAGE conditions, whereas lower molecular weight bands represent fully-glycosylated and core-glycosylated forms of the dissociated monomeric (m) protein. The blots were stripped and reprobed with a mouse monoclonal anti-GAPDH antibody to control for protein loading. B, ratios of fully-glycosylated protein (monomer and dimer)/total NHE6 protein (fg/total) were quantified by densitometry of X-ray films exposed in the linear range and analyzed using ImageJ software. Values represent the mean ± S.D. of three different experiments. Significance was determined by two-way ANOVA with a post hoc Tukey test. The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Population means of the NHE6 variants are significantly different (F value = 26.4, p value = 1.5 × 10−16). Population means as a function of time are significantly different (F value = 48.6, p value = 1.9 × 10−19). § indicates significance (p < 0.01) of the means of NHE6 variants within a cluster relative to the 12-h time point. Asterisks indicate significance (★, p < 0.05, and ★★, p < 0.01) of the means between clusters of NHE6 variants at the indicated time points.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Molecular Weight, Control, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the protein stability of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT of CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points and lysed, and equal amounts of protein (20 μg) were analyzed by Western blotting using a mouse monoclonal anti-HA antibody. Blots were reprobed with a mouse monoclonal anti-GAPDH antibody to control for loading. Blots are representative images from four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of proteasomal and lysosomal inhibitors on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points in the presence of diluent (DMSO) and the proteasomal inhibitor MG-132 (40 μm) or the lysosomal inhibitor leupeptin/pepstatin (LeuP, 100 μg/ml). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of three separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of the lysosomotropic agent chloroquine on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide for the indicated time points in the presence of diluent (H2O) or chloroquine (CQ, 500 μm). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular detection of NHE6 variants in recycling endosomes in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were incubated with the recycling endosomal marker Alexa Fluor 488–conjugated transferrin (Tf-AF488, 10 μg/ml) for 45 min, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and Tf-AF488 channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with Tf-AF488 as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 6479.8, p value = 5.6 × 10−9), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Incubation, Marker, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular localization of certain CS variants in the endoplasmic reticulum in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were immunostained for endogenous CANX, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and CANX channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with CANX as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 3012.9, p value = 1.7 × 10−10), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the functional properties of NHE6 variants. A, biochemical determination of plasma membrane trafficking of NHE6GFP WT or CS-linked variants using a cell-surface biotinylation assay. Cell-surface proteins were labeled with N-hydroxysulfosuccinimidyl–SS–biotin in AP-1 cells expressing the NHE6GFP constructs after 48 h. Total-cell lysates (left panel; protein loading ranged from 10 to 50 μg of protein per sample as indicated below the blot) and biotinylated fractions (right panel; representing 20–100% of plasma membrane proteins extracted per sample) were examined by Western blotting with polyclonal anti-GFP and monoclonal anti-GAPDH antibodies. Representative blots from three experiments are shown. B and C, surface expression and endocytosis of external triple flag tag–labeled NHE6 (3FNHE6HA) constructs in transiently transfected (48 h) AP-1 cells using a cell-based ELISA. Mean intensity fluorescence (M.I.F.) units were determined as a function of the cellular protein concentration and then normalized as percentage (M.I.F. units for WT (100%): 25,100 ± 6,348, n = 4). The surface expression of each construct at time 0 min (before the start of internalization) is charted in B (n = 3–4 experiments). Significance from WT-expressing cells was determined using a one-way repeated measures ANOVA (F value = 463.3, p value = 0.0022), with a post hoc Dunnett's test; *, p < 0.05. Percentage internalization of NHE6 constructs normalized to the zero time point are presented in C and represent the mean ± S.D. (n = 3–4 experiments). The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q, and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Significance from WT cells at the 5- and 15-min time points was determined using a one-way ANOVA (F value = 9.43, p value = 4.48 × 10−5), with a post hoc Tukey test; ★, p < 0.05. D, transferrin uptake in HeLa cells transiently transfected (48 h) with GFP or NHE6GFP constructs. The initial uptake (5 min) of Alexa 633–conjugated transferrin (Tf-AF633) was measured in 1 × 104 GFP-positive HeLa cells per experiment by flow cytometry (M.I.F. units for GFP control: 10,204 ± 1554, n = 4). Data were normalized as a percentage and displayed as percent change from GFP control cells. Significance from control cells was determined using a one-way repeated measures ANOVA (F value = 320.7, p value = 3.8 × 10−4), with a post hoc Dunnett's test; ★★, p < 0.001. E, recycling endosomal pH (pHe) was measured in AP-1 cells in the absence or presence of transiently transfected (48 h) NHE6ChFP constructs by fluorescence ratio image analysis of the internalized pH-sensitive probe FITC-conjugated human transferrin (Tf–FITC). Data represent the average endosomal pHe per cell pooled from three separate experiments (8–12 cells per construct/experiment; n = 24–36). Significance was determined by one-way ANOVA (F value = 40.02, p value = 0), with a post-hoc Tukey test; ★★, p < 0.001. Data in B, D, and E are plotted as box charts, with the central white square indicating the mean; the box representing the S.E.; and the error bars showing the S.D. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Functional Assay, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Labeling, Expressing, Construct, Western Blot, FLAG-tag, Transfection, In-Cell ELISA, Fluorescence, Protein Concentration, Flow Cytometry, Control

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Effect of pre B cell leukemia transcription factor 1 (PBX1) and PBX2 on valosin‐containing protein (VCP) expression evaluated by siRNA and RT‐PCR analysis on non‐small cell lung cancer (NSCLC) cell lines PC14 and A549. (A) Relative amounts of PBX1 and VCP mRNA to GAPDH mRNA were shown. Decrease in PBX1 expression level by siRNA did not affect the amount of VCP mRNA. (B) Relative amounts of PBX2 and VCP mRNA were shown. The amount of VCP mRNA significantly reduced when PBX2 expression level decreased. *P < 0.05 by Student's test.

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Analysis of pre B cell leukemia transcription factor 2 (PBX2) binding to the 5′‐flanking region of the human valosin‐containing protein (VCP) gene. ChIP assay was performed using non‐small cell lung cancer (NSCLC) cells. Input DNA sample with (lane A) or without (lane B) the addition of anti‐PBX2 antibody was immunoprecipitated with anti‐PBX2 (lane C) or preimmune IgG serum (lane D). Precipitated DNA fragments were PCR‐amplified with primers specific for the VCP promoter region. PCR products were separated on 2% agarose gel and stained with ethidium bromide. Positive band in the lane of the sample precipitated with anti‐PBX2 antibody (lane A) was seen at the same size with those of input DNA (lanes C and D). M; molecular weight marker.

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Binding Assay, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Pre B cell leukemia transcription factor 2 (PBX2) overexpression up‐regulated luciferase activity of valosin‐containing protein (VCP) promoter in non‐small cell lung cancer (NSCLC) cells of A549 (A) and PC14 (B). The luciferase acivity showed a significant decrease with the transfection of the plasmid mutated or deleted at four PBX2 binding sites. When PBX2 expression plasmid was co‐transfected, the reporter activity significantly increased. Bars represent mean ± SE of at least three independent experiments. *Significant difference (P < 0.05).

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Over Expression, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Expressing

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Comparison of pre B cell leukemia transcription factor 2 (PBX2) protein level determined by Western blot (WB) and immunohistochemistry (IHC). (A) Amount of PBX2 and histone‐H3 proteins in tissues from non‐small cell lung cancer (NSCLC) was determined by WB. (B) The relative amount of PBX2 protein in cases with high and low expression by IHC.

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Comparison, Western Blot, Immunohistochemistry, Expressing

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Verification of the specificity of anti‐pre B cell leukemia transcription factor 2 (PBX2) and anti‐valosin‐containing protein (VCP) antibodies. Immunohistochemistry with pre‐absorbed anti‐PBX2 (A), non‐absorbed anti‐PBX2 (B), pre‐absorbed anti‐VCP (C), non‐absorbed anti‐VCP (D) are shown. × 200.

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Immunohistochemistry

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Low and high expression of pre B cell leukemia transcription factor 2 (PBX2) by immunohistochemistry. Avidin‐biotin‐peroxidase complex method, ×200.

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing, Immunohistochemistry, Avidin-Biotin Assay

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Relationship between PBX2 expression and clinicopathologic factors in 206 patients with non‐small cell lung cancer

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing, Adjuvant

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Univariate analysis of clinicopathologic factors for disease‐free and overall survival in 206 patients with non‐small cell lung cancer

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing, Adjuvant

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Multivariate analysis of clinicopathologic factors for disease‐free and overall survival in 206 patients with non‐small cell lung cancer

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Relationship between PBX2 expression and clinicopathologic factors in 173 non‐small cell lung cancer patients with non‐adjuvant therapy

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Disease‐free and overall survival (DFS and OS) rates of patients with high and low expression of pre B cell leukemia transcription factor 2 (PBX2) and valosin‐containing protein (VCP) in non‐small cell lung cancer patients without adjuvant therapy. Significant difference was observed between patients with high and low expression of PBX2 (A and B) and VCP (C and D) for DFS and OS.

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing, Adjuvant

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Univariate analysis of clinicopathologic factors for disease‐free and overall survival in 173 non‐small cell lung cnacer patients with non‐adjuvant therapy

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Multivariate analysis of clinicopathologic factors for disease‐free and overall survival in 173 non‐small cell lung cancer patients with non‐adjuvant therapy

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing

Journal: Cancer Science

Article Title: Prognostic significance of pre B cell leukemia transcription factor 2 (PBX2) expression in non‐small cell lung carcinoma

doi: 10.1111/j.1349-7006.2009.01156.x

Figure Lengend Snippet: Disease‐free and overall survival (DFS and OS) rates of patients with high and low expression of pre B cell leukemia transcription factor 2 (PBX2) and VCP at Stage I disease of pathologic tumor‐node‐metastasis (pTNM) classification. Significant difference in survival was not observed between patients with high and low PBX2 expression (A and B), but significant difference was observed between high and low VCP expression for DFS and OS (C and D).

Article Snippet: To evaluate the specificity of primary antibodies, the pre‐absorption of anti‐PBX2 and anti‐VCP antibodies with the peptide of PBX2 and VCP (Santa Cruz Biotechnologies; 1:5), respectively, were performed.

Techniques: Expressing

Journal: bioRxiv

Article Title: Endosomal trafficking is required for glycosylation and normal maturation of the Alzheimer’s-associated protein sorLA

doi: 10.1101/2020.07.12.199885

Figure Lengend Snippet: (A) WB analysis of lysate and conditioned medium from SH-SY5Y cells stably transfected with sorLA WT . (B) Schematic representation of proposed N -glycosylation variants for two molecular species of sorLA (mature: arrow open, and immature: arrow filled) in lysates and one in medium (mature shed: arrow red). The high molecular weight form (upper band; sorLA-mat) carries a mixture of mature complex-type (C) and high-mannose (H) N -glycans, whereas the low molecular weight form (lower band; sorLA-im) only carries immature high-mannose N -glycans. (C) Cell surface proteins from SH-SY5Y cells expressing exogenous sorLA WT were labeled with biotin and precipitated using Streptavidin coated beads. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA and calnexin (ER marker). (D) Eluates of biotinylated cell surface proteins from SH-SY5Y and HEK293 cells expressing exogenous sorLA WT treated (+) or not (-) with neuraminidase after precipitation using Streptavidin coated beads as indicated. (E, F) SH-SY5Y cells expressing either sorLA WT or sorLA KKLN were subjected to a [ 35 S] pulse-chase protocol in order to follow the maturation over time. Cells were harvested at the indicated time points, and sorLA protein from lysate and medium was immunoprecipitated, separated by SDS-PAGE analysis, and visualized by radiofluorography. Signals in WB for lysates for sorLA-mat and sorLA-im are indicated with white and black arrowheads, and for shed sorLA in medium with a red arrowhead, respectively.

Article Snippet: Recognition of glycosylated sorLA by lectins was carried out applying biotinylated lectins (10 μg/mL; Biotinylated Lectin Kit I and Biotinylated SNA, Vector Laboratories) and peroxidase-conjugated streptavidin (Calbiochem) diluted 1:1500.

Techniques: Stable Transfection, Transfection, Molecular Weight, Expressing, Labeling, SDS Page, Marker, Pulse Chase, Immunoprecipitation

Journal: bioRxiv

Article Title: Endosomal trafficking is required for glycosylation and normal maturation of the Alzheimer’s-associated protein sorLA

doi: 10.1101/2020.07.12.199885

Figure Lengend Snippet: (A) Schematic representation of classical N -glycan maturation in the secretory pathway. Tunicamycin blocks initiation of protein N -glycosylation, whereas MGAT1 is required for conversion of high-mannose glycans and ST3Gal sialyltransferases for capping of the glycans as indicated. Designations for monosaccharides follow the Consortium for Functional Glycomics. (B) SH-SY5Y cells expressing exogenous sorLA WT were treated with tunicamycin (1 μg/mL) for 24 h prior to biotinylation of cell surface proteins. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA. C) SH-SY5Y cells expressing exogenous sorLA WT were subjected to a 40 min [ 35 S]-labeling and chased for the indicated time points in absence or presence (+) of tunicamycin. SorLA proteins from cell lysates and medium were immunoprecipitated, and subsequent analyzed by SDS-PAGE and radio-fluorography. (D) Wild type CHO (CHO WT ) or CHO cells with targeted deletion of Mgat1 (CHO KOmgat1 ), St3gal4 and St3gal6 (CHO KOst3gal4/6 ), or St3gal3, St3gal4 , and St3gal6 (CHO KOst3gal3/4/6 ) were transfected with sorLA WT and shedding efficiency determined by WB analysis of cell lysates and conditioned medium. (E) Quantification of shed sorLA from three independent experiments as illustrated in panel D. Signals for sorLA-mat (upper band) and sorLA-im (lower band) in lysates are indicated with white and black arrowheads, respectively, while shed sorLA represented by a red arrow. The grey arrowhead indicates migration of the nascent sorLA protein in lysate with no N -glycans. (F) SH-SY5Y cells expressing exogenous sorLA WT were subjected to surface biotinylation (time 0 h) and treatment with neuraminidase (Neu) to remove sialic acids on surface located sorLA. Following cultivation at 37°C for 6 h total cell lysates were subjected to precipitation on Streptavidin beads (time 6 h) and analyzed by WB for detection of sorLA species. Note that surface biotinylated sorLA at time 0 h after neuraminidase treatment co-migrates with the lower migrating sorLA-im, and at time 6 h regains co-migration with the sorLA-mat upper band. To demonstrate that the surface biotinylated sorLA species detectable after 6 h indeed had regained sialic acids the streptavidin precipitate was subjected to neuraminidase again (Neu post-treatment, beads), and in agreement with this a clear shift in mobility was observed.

Article Snippet: Recognition of glycosylated sorLA by lectins was carried out applying biotinylated lectins (10 μg/mL; Biotinylated Lectin Kit I and Biotinylated SNA, Vector Laboratories) and peroxidase-conjugated streptavidin (Calbiochem) diluted 1:1500.

Techniques: Functional Assay, Expressing, SDS Page, Labeling, Immunoprecipitation, Transfection, Migration

Journal: bioRxiv

Article Title: Endosomal trafficking is required for glycosylation and normal maturation of the Alzheimer’s-associated protein sorLA

doi: 10.1101/2020.07.12.199885

Figure Lengend Snippet: (A) Schematic representation of the action of dynasore (DYN) and chlorpromazine (CPZ) that block dynamin-dependent endocytosis and the conversion from coated vesicles to endosomes during clathrin-coated internalization, respectively. (B) Confocal microscopy displaying the localization of sorLA WT in SH-SY5Y cells treated with DYN (center) or CPZ (bottom) for 24 h or non-treated cells (top). The large white arrowhead indicates perinuclear localization of sorLA WT in non-treated cells. The small white arrowheads indicate punctuate (i.e. endosome-like vesicular) localization of sorLA WT in cells treated with CPZ (scale bar = 10 μm). (C) SH-SY5Y cell expressing exogenous sorLA WT were treated with DYN or CPZ for 24 h prior to biotinylation of surface proteins. Total lysates and biotinylated proteins were analyzed using SDS-PAGE and Western blotting for sorLA. The signals for the mature and the immature sorLA WT in samples from total lysates were determined by densitometric scanning of Western blots from three independent experiments and.the level of maturation (expressed as the level of the upper variant relative to the sum of both variants) is shown in bar graph, as mean ± S.E.M. (D) Bar graph representing the amount of sorLA WT on the cell surface of both variants relative to the total level of sorLA on the plasmamembrane on cells with no treatment.

Article Snippet: Recognition of glycosylated sorLA by lectins was carried out applying biotinylated lectins (10 μg/mL; Biotinylated Lectin Kit I and Biotinylated SNA, Vector Laboratories) and peroxidase-conjugated streptavidin (Calbiochem) diluted 1:1500.

Techniques: Blocking Assay, Confocal Microscopy, Expressing, SDS Page, Western Blot, Variant Assay

Journal: bioRxiv

Article Title: Endosomal trafficking is required for glycosylation and normal maturation of the Alzheimer’s-associated protein sorLA

doi: 10.1101/2020.07.12.199885

Figure Lengend Snippet: (A) WB analysis of lysates and medium from transfected CHO cells that express sorLA WT , sorLA FANSHY or sorLA ΔCD using a polyclonal antibody against the luminal receptor domain. (B) Cells treated with the TACE inhibitor Gm6001 (+) shows strongly reduced shed sorLA in the medium, accompanied by accumulation of the upper variant in lysates, most prominently observed for cells expressing sorLA ΔCD as these undergo much faster processing than sorLA WT . (C) SH-SY5Y cells expressing sorLA ΔCD lacking the cytosolic tail were subjected to a pulse-chase protocol in order to follow maturation and shedding over an 8 h interval. Cells and medium were harvested at the indicated time points, and sorLA ΔCD proteins were immunoprecipitated and subsequent analyzed by SDS-PAGE and radiofluorography. Quantification of mature (white) and immature (black) sorLA ΔCD from lysates is shown from three independent experiments is shown below. (D) WB analysis of lysates and medium from SH-SY5Y cells expressing sorLA ΔCD indicates loss of mature variant in the lysate caused by shedding of the high-molecular variant. (E) Cell surface proteins from SH-SY5Y cells expressing sorLA FANSHY were labeled with biotin and precipitated using Streptavidin coated beads. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA and calnexin. (F) Lysates from SH-SY5Y cells expressing sorLA FANSHY were enzymatically digested with Endo H, PNGase F, and either neuraminidase (Neu) alone or in combination with O -glycosidase. The homogenates were subsequently separated by SDS-PAGE and analyzed by WB.

Article Snippet: Recognition of glycosylated sorLA by lectins was carried out applying biotinylated lectins (10 μg/mL; Biotinylated Lectin Kit I and Biotinylated SNA, Vector Laboratories) and peroxidase-conjugated streptavidin (Calbiochem) diluted 1:1500.

Techniques: Transfection, Variant Assay, Expressing, Pulse Chase, Immunoprecipitation, SDS Page, Labeling

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: 1A . Comparison of relative expression for BAMBI mRNA in kidneys from wild type ( BAMBI +/+ ), heterozygous ( BAMBI +/− ), or BAMBI-deleted ( BAMBI −/− ) mice expressed as fold change relative to BAMBI +/+ controls. 1B . Western blot for BAMBI protein in kidney lysates from BAMBI +/− , BAMBI −/− , as well as BAMBI +/+ mice and normal human kidney. For localization of BAMBI, lane 1 shows lysate from HEK 293 cells overexpressing BAMBI. Please note that the human BAMBI travels at a molecular weight around 28–29 kDa as compared to the mouse BAMBI with a molecular weight of 26–27 kDa. Tubulin mRNA is shown as a loading control for the BAMBI +/− and BAMBI −/− kidney lysates.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Comparison, Expressing, Western Blot, Molecular Weight, Control

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: Comparison of relative mRNA levels for BAMBI in isolated glomerular and tubular fractions from murine and human kidneys. Levels of glomerular-specific podocyte marker genes, podocin or WT-1, respectively, indicate their enrichment in the glomerular preparation and their low levels in the tubular preparations. Values are means +/− SEM of 3–4 experiments for each set.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Comparison, Isolation, Marker

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: 3A . BAMBI mRNA levels as copy numbers (means +/− SEM) in murine mesangial cells (mMC; n = 4), murine glomerular endothelial cells (mGEC; n = 3) or human umbilical vein endothelial cells (HUVEC; n = 14). 3B . Western blot for BAMBI in glomeruli isolated from mouse kidney, in cultured mGECs and HUVECs. For comparison the position of murine BAMBI overexpressed in HEK 293 cells is shown. Blot for GAPDH is shown as loading control. 3C . In situ hybridization for BAMBI in human renal blood vessel and in human glomerulus. Positive signals are only obtained with the antisense, but not with the sense probe and localize to endothelial cells in the blood vessel and in glomerular capillary loops.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Western Blot, Isolation, Cell Culture, Comparison, Control, In Situ Hybridization

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: 4A . Immunofluorescence staining for BAMBI in frozen sections from kidneys of BAMBI +/+ and BAMBI −/− mice (10× and 40× magnification). At low power, glomeruli are positive for BAMBI staining (green) in the BAMBI +/+ kidneys, while only background fluorescence is seen in kidney from BAMBI −/− mice. At high magnification the staining for BAMBI is seen in a glomerulus of the BAMBI +/+ kidney, whereas only background fluorescence is apparent in the glomerulus from BAMBI −/− mice. 4B . Immunofluorescence staining for BAMBI (red) and synaptopodin (green) as a podocyte-specific marker in frozen sections from human kidney with an endothelial distribution and lack of colocalization (arrows) with synaptopodin. 4C . Double immunofluorescence staining for BAMBI (red) and CD31 (green) as an endothelial marker and the merged picture in a glomerulus at high magnification from a frozen section from a kidney of a BAMBI +/+ mouse. The specific co-staining for BAMBI and CD31 is apparent in the merged picture as yellow/orange on the luminal aspects of the glomerular capillaries, as indicated by arrows. 4D . Double immunofluorescence staining for BAMBI (red) and CD31 (green) and the merged picture in a renal medium-sized artery at high magnification from a frozen section from a kidney of a BAMBI +/+ mouse The specific co-staining for BAMBI and CD31 is apparent in the merged picture as yellow/orange on the luminal aspects of the vascular endothelium. 4E . Double immunofluorescence staining of a section of an artery from a BAMBI −/− mouse. Only some faint, non-specific, red background staining is apparent for BAMBI in the BAMBI −/− section, whereas endothelial CD31 staining remains strong. 4F . Double immunofluorescence staining in human kidney showing positive staining for BAMBI and CD31 in the endothelium of a mid-sized artery with colocalization as shown in the merged picture (yellow/orange).

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Fluorescence, Marker, Double Immunofluorescence Staining

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: Effects of TGFβ or LPS on the mRNA levels for BAMBI in murine mesangial cells (mMC) and murine glomerular endothelial cells (mGEC) and human umbilical vein endothelial cells (HUVEC). Cells were incubated under control conditions or with the addition of TGFβ (5 ng/ml) or LPS (1 µg/ml) for 6 and 24 hours. Data for mRNA are corrected for actin expression and calculated as fold change compared to the respective untreated controls. Values represent means +/− SEM from 3–7 independent series of experiments with TGFβ and of 3–13 series with LPS. Asterisks indicate p<0.05 as compared to the respective controls.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Incubation, Control, Expressing

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: Fold change values for BAMBI mRNA in response to TGFβ and LPS.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques:

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: Localization of ARE sequences in the 3′untranslated region of the BAMBI gene.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques:

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: 6A . Effect of actinomycin on the mRNA levels for BAMBI in murine glomerular endothelial cells (mGEC). BAMBI mRNA levels were compared in mGECs after up to 4 hours of incubation in control medium (CTRL) or in medium with addition of 5ug/ml of actinomycin to inhibit de novo mRNA synthesis. All values are expressed as percentage of the respective zero-hour time controls. Values are means +/− SEM of three series of experiments. Asterisk refer to p<0.01 for actinomycin versus controls. 6B . Effects of cycloheximide on the mRNA levels for BAMBI in mGEC. Cells were incubated for the times indicated under control conditions or in the presence of cycloheximide (1mg/ml) prior to RNA extraction. Values for mRNA of BAMBI are expressed as percentage of the respective zero hour time controls. Values are means +/− SEM of three series of experiments. Asterisk refer to p<0.01 for cycloheximide versus controls. 6C . Effects of 6 hours of cycloheximide treatment on the mRNA levels for BAMBI in mMCs, mGECs, and HUVECs. Values are means +/− SEM from 2–4 series of experiments for each cell type.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Incubation, Control, RNA Extraction

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: 7A . Representative Western blot and densitometry analysis for BAMBI in HUVECs overexpressing BAMBI (lentiviral system). Cells were cultured in 10% FBS or 0.2% FBS for 24 hours prior to protein extraction. GAPDH is shown as loading control. 7B . For densitometry analysis, normalized relative intensities were calculated from band intensity values obtained using the NIH Image J program. Data are means +/− SEM of relative BAMBI∶GAPDH ratios (normalized to 10% FBS control cells) for two independent experiments.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Western Blot, Cell Culture, Protein Extraction, Control

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: 8A . Western blot and densitometry for BAMBI protein in BAMBI-overexpressing HUVECs. Cells were serum-starved (0.2% FBS) for 24 hours in the absence (CTRL) or presence of bafilomycin; 3MA; MG132 or epoxomicin for 24 hours at the concentrations described in Methods . Normalized relative intensities were calculated from band intensity values obtained using the NIH Image J program. Data are means +/− SEM of BAMBI∶GAPDH ratios (normalized to 0.2% FBS controls) for two independent experiments. 8B . Western blots for BAMBI, GAPDH, and ubiquitinated proteins in 293T cells infected with BAMBI-lentivirus cultured in 10% FBS and treated with bafilomycin or epoxomicin for 8 hours. After staining the Western blot for BAMBI and GAPDH, the stripped membrane was stained with an antibody for ubiquitinated proteins. While the proteasomal inhibitor epoxomicin caused a marked accumulation of ubiquitinated proteins, consistent with inhibition of their proteasomal degradation, bafilomycin did not detectably influence ubiquitinated protein degradation and retention.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Western Blot, Infection, Cell Culture, Staining, Membrane, Inhibition

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: Western blot for BAMBI, GAPDH, and LC3 in BAMBI overexpressing HUVECs cultured in 10% FBS and treated with vehicle (CTRL), rapamycin, bafilomycin or bafilomycin plus rapamycin for 24 hours. Rapamycin alone reduces the BAMBI band, whereas bafilomycin alone and even in combination with rapamycin enhances it. Rapamycin causes a shift from the LC3-I to the LC3-II band, and bafilomycin alone enhances both LC3 bands. Bafilomycin does not, however, prevent the shift from LC3-I to LC3-II induced by rapamycin.

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Western Blot, Cell Culture

Journal: PLoS ONE

Article Title: BAMBI Is Expressed in Endothelial Cells and Is Regulated by Lysosomal/Autolysosomal Degradation

doi: 10.1371/journal.pone.0012995

Figure Lengend Snippet: 10A . Confocal images for immunofluorescence staining of BAMBI (red), LC3 (green) and DAPI (blue) in HUVECs with BAMBI overexpression. Cells were cultured in 10% or 0.2% FBS for 24 hours prior to fixation and immunofluorescence staining. Note marked perinuclear punctate staining for BAMBI and diffuse and occasional punctate staining for LC3 in cells maintained with 10% FBS with hardly any merge of signals. After 24 hours of culture in 0.2% FBS there is an overall reduction in the punctate red staining for BAMBI and a marked increase in punctate green staining for LC3, resulting in some clearly discernable colocalization and merge to an orange punctate signal in some areas. For quantification of the colocalization please refer to Results . 10B . Confocal images of immunofluorescence for DAPI (blue), BAMBI (red), and LC3 (green) staining in BAMBI overexpressing HUVECs cultured in 10% FBS and treated with vehicle, rapamycin, bafilomycin or bafilomycin plus rapamycin for 24 hours. Rapamycin alone caused the diffuse LC3 staining to take on a distinct punctate appearance, which was further enhanced by bafilomycin alone or in combination with rapamycin. Rapamycin alone slightly reduced the immunofluorescence appearance of BAMBI, whereas bafilomycin alone and especially in combination with rapamycin caused a marked increase in the BAMBI-positive puncta. For quantification please refer to Results .

Article Snippet: The primary antibodies used in these studies were as follows: goat polyclonal anti-mouse BAMBI (R&D Systems), rat monoclonal anti-mouse CD31 (Santa Cruz Biotechnology), mouse monoclonal anti-human BAMBI (Abnova), mouse monoclonal anti-rat synaptopodin (Progen, Heidelberg, Germany), rabbit polyclonal anti-human synaptopodin (Sigma-Aldrich), rabbit polyclonal anti-human EEA1 (Cell Signaling Technology), mouse monoclonal anti-bovine ubiquitin (Zymed laboratories), rabbit polyclonal anti-human LC3 (Novus Biologicals), and mouse monoclonal anti-rabbit GAPDH (Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Over Expression, Cell Culture