Journal: The Journal of Neuroscience

Article Title: Interdependent Conductances Drive Infraslow Intrinsic Rhythmogenesis in a Subset of Accessory Olfactory Bulb Projection Neurons

doi: 10.1523/JNEUROSCI.2520-15.2016

Figure Lengend Snippet: Expression of BK and CaV2.3 channels in AOB mitral cells. A–C, Confocal laser-scanning microscopy images of the mouse AOB (parasagittal cryosections). Immunofluorescence labeling (green) of CaV2.3 (A), the BK channel subunit α-1 (Sloα1; B), and the BK channel subunit β-4 (Sloβ4; C). Nuclei are counterstained with DRAQ5 (red). Dashed lines indicate both the lateral olfactory tract (LOT) and the glomerular layer (GL) in Aiii, Biii, and Ciii. Representative single optical section images show an overview of the AOB (Ai, Bi, Ci) and a high-magnification view (Aii, Bii, Cii) of the mitral cell layer (MCL). Note the robust labeling of cells in the MCL as well as the absence of immunofluorescence following peptide preadsorption (Aiii, Biii, Ciii; no DRAQ5 counterstaining). D, E, Post hoc immunolabeling of previously recorded intrinsically rhythmic neurons. Confocal images of AOB cryosections containing biocytin-filled iAMC somata and proximal dendrites (visualized by Alexa Fluor 488/633 streptavidin conjugate). Sections were counterstained against the BK channel α-1 (D) and β-4 (E) subunits, respectively. Merged images (Diii, Eiii) reveal BK expression in these representative iAMCs. Div, Eiv, Western blot analysis of total olfactory bulb (right lanes) and posterior brain (left lanes) protein extracts. Immunoblots were probed with anti-Sloα1 (D) and anti-Sloβ4 (E) antibodies, respectively. Expected band size is indicated above both blots. d, Dorsal; FC, frontal cortex; v, ventral.

Article Snippet: Primary antibodies were anti-Slo1 (K Ca 1.1, 1:500; Alomone Labs), anti-Sloβ4 (1:100; Alomone Labs), and anti-Ca V 2.3 (1:100; Alomone Labs).

Techniques: Expressing, Confocal Laser Scanning Microscopy, Immunofluorescence, Labeling, Immunolabeling, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Membrane colocalization and overlapping between α3β4 nAChR (red) and DBH (green) after secretion stimulus: in each image we individually selected several regions of interest (ROIs) ( a , b ) and proceeded to analyze them using ImageJ JACoP complement (See ), obtaining the values of the Pearson’s and Manders coefficients, whose averages are shown in , as well as their corresponding scatterplot graphs, whose examples are shown ( d ). The averaged Pearson’s coeficients for 50% threshold images show a significant colocalization between endogenous α3β4 nAChRs structures and the DBH sites in both stimulation conditions to those obtained for randomly simulated α3β4 nAChRs structures ( ( c )). Furthermore, the averaged coefficients obtained for the stimulus with ACh are significantly higher than those obtained when stimulating with a high K + ( p value *** < 0.0001; ). ( d ) A scatterplot or fluorogram for pixel colocalization of the red channel (AChR structures) and green channel (DBH) comparing three examples of the images obtained in each of the three analyzed conditions. Bars represent 1 µm.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques: Membrane

Journal: International Journal of Molecular Sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Parameters used in the simulation of buffered calcium diffusion in a conical domain.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques: Diffusion-based Assay, Concentration Assay, Binding Assay

Journal: Cell Stem Cell

Article Title: Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming

doi: 10.1016/j.stem.2017.10.013

Figure Lengend Snippet:

Article Snippet: The following antibodies were used with indicated dilution: ICAM1-biotin conjugate (1:100, eBioscience, #13-0541), CD44-allophycocyanin (APC) conjugate (1:300, eBioscience, #17-0441), E-CADHERIN-eFluor 660 (1:300, eBioscience, #50-3249-82), MEFSK4-biotin conjugate (1:100, Miltenyi, 130-101-875), CD47-biotin conjugate (1:100, BioLegend, 127505), CD73-Alexa Fluor 647 (1:300, BD Biosciences, 561543) and CD104-APC (1:300, Miltenyi, 130-106-924) and Strepdavidin PE-Cy7 (1:1500, eBioscience, #25-4317).

Techniques: Recombinant, Software

Journal: Scientific Reports

Article Title: N -Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

doi: 10.1038/srep33507

Figure Lengend Snippet: ( a ) Schematic diagram of potential N -glycosylation sites on the WT and mutational integrin α5 subunits (WT; S3–5; S3–5,10–14; S3–5,10; S3–5,11; S3–5,12; S3–5,13 and, S3–5,14). Putative N -glycosylation sites (N84Q, N182Q, N297Q, N307Q, N316Q, N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q) are indicated by solid triangles, and point mutations are indicated by hollow triangles. ( b ) The integrin α5 mutant cells expressed equal α5β1 and EGFR levels on the cell surface, compared with the WT cells. The stable cell lines were established as described under the “Methods” section. The expression levels of both α5β1 and EGFR were analyzed by flow cytometry. The IgG was used as a control. ( c ) The α5 mutant cells exhibited abilities for cell spreading that were comparable to those of WT. Cells were detached and then replated on the FN-coated dishes. After incubation for 20 min, cells were fixed and the representative images were taken. The percentages of rounded, spread, and elongated cells were statistically analyzed ( right bottom panel, n = 9). Scale bar , 120 μm. ( d ) The S3–5,11 cells exhibited inhibitory ability for cell growth that was comparable to that of the S3–5,10–14 versions. After starvation for 24 h, cells were supplied with DMEM containing 10% FBS, and then cell numbers were counted and statistically analyzed at indicated times ( n = 3 individual experiments). All values are reported as the means ± S.E. ( error bars ), Student’s t -test; n.s, not significant ( p > 0.05 ); ** p < 0.01; *** p < 0.001.

Article Snippet: The experiments were performed using the following antibodies: monoclonal antibodies (mAbs) against integrin α5 (610634) and integrin β4 (611233) were obtained from BD Biosciences; the mAbs against human integrin α5β1 (MAB1999) and integrin β4 (MAB2059) were from Millipore; the mouse pAbs to EGFR (Sc-120), integrin α3 (Sc-6592), or integrin α3 (Sc-32237) and the rabbit pAbs to integrin β4 (Sc-9090) or EGFR (Sc-03) were from Santa Cruz Biotechnology; rabbit mAbs to EGFR (catalog no. 4267), p-EGFR (catalog no. 3777), ERK1/2 (catalog no. 9102), p-ERK1/2 (catalog no. 4370), AKT (catalog no. 9272), and p-AKT (catalog no. 4060) were from Cell Signaling Technology; and, the mAb against α-tubulin was from Sigma.

Techniques: Mutagenesis, Stable Transfection, Expressing, Flow Cytometry, Incubation

Journal: Scientific Reports

Article Title: N -Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

doi: 10.1038/srep33507

Figure Lengend Snippet: ( a ) The expression levels of phospho-EGFR and its downstream signaling were down-regulated in the S3–5,11 cells. Cell lysates from the indicated cells were subjected to western blot ( WB ) with indicated antibodies ( top panel ). The relative ratios (phospho-EGFR, phospho-ERK, and phospho-AKT versus EGFR, ERK, and AKT, respectively) were statistically analyzed ( bottom panel, n = 3 individual experiments). ( b ) The interaction between integrin α5 and EGFR was increased in the S3–5,11 cells. The indicated cell extracts were immunoprecipitated ( IP ) with anti-GFP agarose, followed by anti-EGFR and α5 antibodies for WB ( top panels ). The whole cell extracts were also subjected to WB using indicated antibodies where α-tubulin was used to check equal loading ( bottom panels ; as an input). All values are reported as the means ± S.E. ( error bars ), Student’s t -test; *** p < 0.001.

Article Snippet: The experiments were performed using the following antibodies: monoclonal antibodies (mAbs) against integrin α5 (610634) and integrin β4 (611233) were obtained from BD Biosciences; the mAbs against human integrin α5β1 (MAB1999) and integrin β4 (MAB2059) were from Millipore; the mouse pAbs to EGFR (Sc-120), integrin α3 (Sc-6592), or integrin α3 (Sc-32237) and the rabbit pAbs to integrin β4 (Sc-9090) or EGFR (Sc-03) were from Santa Cruz Biotechnology; rabbit mAbs to EGFR (catalog no. 4267), p-EGFR (catalog no. 3777), ERK1/2 (catalog no. 9102), p-ERK1/2 (catalog no. 4370), AKT (catalog no. 9272), and p-AKT (catalog no. 4060) were from Cell Signaling Technology; and, the mAb against α-tubulin was from Sigma.

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: N -Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

doi: 10.1038/srep33507

Figure Lengend Snippet: ( a ) Schematic diagram of a potential N -glycosylation mutational integrin α5 subunit (WT; S3–5; D11; and, S3–5,11). ( b , c ) Comparison of expression levels of α5β1 and EGFR on the cell surface ( b ) and cell spreading ( c ) among the WT; D11; and S3–5,11 cells. The stable rescued-HeLa cell lines were established as described under the “Methods” section. The expression levels of both α5β1 and EGFR on the cell surface were analyzed via flow cytometry ( b ). The cell-spreading abilities of these cells were analyzed, as described in ( n = 3 individual experiments). The percentages of rounded and spread cells were statistically analyzed ( c , bottom panel, n = 9). Scale bar , 120 μm. ( d ) The site-11 N -glycosylation suppressed cell growth. The abilities for cell growth were compared among the parent (Par.); integrin α5-knock-out (KO); WT; S3–5; D11; and S3–5,11 HeLa cells, as described in ( n = 3 individual experiments). ( e ) The expression levels of phospho-EGFR and its downstream signaling were significantly increased in the D11, but not in the S3–5,11 cells compared with the WT. Left panel , WB pattern; right panel , quantitative analysis ( n = 3 individual experiments). All values are reported as the means ± S.E. ( error bars ), Student’s t -test; n.s, not significant ( p > 0.05 ); ** p < 0.01; *** p < 0.001.

Article Snippet: The experiments were performed using the following antibodies: monoclonal antibodies (mAbs) against integrin α5 (610634) and integrin β4 (611233) were obtained from BD Biosciences; the mAbs against human integrin α5β1 (MAB1999) and integrin β4 (MAB2059) were from Millipore; the mouse pAbs to EGFR (Sc-120), integrin α3 (Sc-6592), or integrin α3 (Sc-32237) and the rabbit pAbs to integrin β4 (Sc-9090) or EGFR (Sc-03) were from Santa Cruz Biotechnology; rabbit mAbs to EGFR (catalog no. 4267), p-EGFR (catalog no. 3777), ERK1/2 (catalog no. 9102), p-ERK1/2 (catalog no. 4370), AKT (catalog no. 9272), and p-AKT (catalog no. 4060) were from Cell Signaling Technology; and, the mAb against α-tubulin was from Sigma.

Techniques: Expressing, Flow Cytometry, Knock-Out

Journal: Scientific Reports

Article Title: N -Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

doi: 10.1038/srep33507

Figure Lengend Snippet: ( a ) Comparison of complex formations between EGFR-α5 and EGFR-β4 among the WT; D11; and S3–5,11 cells. The indicated HeLa cell extracts were IP with anti-EGFR antibody ( top IP panels ), anti-GFP agarose ( middle IP panels ), or anti-integrin β4 antibody ( bottom IP panels ), and then subjected to WB, reciprocally followed by anti-integrin α5, β4, α3, or EGFR antibodies for detection. The whole cell extracts were also subjected to WB as an “input” using the indicated antibodies ( bottom panels ). ( b ) The WT; D11; and S3–5,11 cells exhibit the same expression level of integrin β4 and α3 on the cell surface. The expression levels of both integrins β4 and α3 were analyzed by flow cytometry. The IgG was used as a control.

Article Snippet: The experiments were performed using the following antibodies: monoclonal antibodies (mAbs) against integrin α5 (610634) and integrin β4 (611233) were obtained from BD Biosciences; the mAbs against human integrin α5β1 (MAB1999) and integrin β4 (MAB2059) were from Millipore; the mouse pAbs to EGFR (Sc-120), integrin α3 (Sc-6592), or integrin α3 (Sc-32237) and the rabbit pAbs to integrin β4 (Sc-9090) or EGFR (Sc-03) were from Santa Cruz Biotechnology; rabbit mAbs to EGFR (catalog no. 4267), p-EGFR (catalog no. 3777), ERK1/2 (catalog no. 9102), p-ERK1/2 (catalog no. 4370), AKT (catalog no. 9272), and p-AKT (catalog no. 4060) were from Cell Signaling Technology; and, the mAb against α-tubulin was from Sigma.

Techniques: Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: N -Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

doi: 10.1038/srep33507

Figure Lengend Snippet: ( a ) The competitive relationship between integrins α5 and β4 to interact with EGFR in HeLa cells. The integrin α5 (GFP-tagged)-overexpressed HeLa stable cell line (OE) was established as described under the “Methods” section. The extracts from integrin α5-knock-out (KO), parent (Par.), and OE HeLa cells were IP with anti-EGFR antibody followed by anti-integrin α5, β4, and EGFR antibodies for WB ( top panels ). ( b ) The interaction patterns between EGFR and integrins β4 or α5 in the WT and D11 HeLa cells spread on FN. The indicated cells were cultured on dishes pre-coated with or without 10 μg/ml FN for 24 h. The resultant cell lysates (as an input; bottom panels ) were directly blotted with indicated antibodies or IP with anti-EGFR antibody ( top panels ) and then blotted with anti-integrin α5, β4, and EGFR antibodies. ( c , d ) Comparison of the phospho-EGFR and phopho-ERK expressions ( c ) and cell proliferation abilities ( d ) between the WT and D11 cells spread on FN or laminin-332 (LN-332). The WT and D11 cells were cultured on dishes pre-coated with or without 10 μg/ml FN or 1 μg/ml LN-332 for 24 h. The resultant cell lysates were subjected to WB with indicated antibodies (c , top panels ), the relative ratios were statistically analyzed (c , bottom panels, n = 3 individual experiments). The analysis of cell growth was performed as described in the legend to , the related cell numbers at 72 h were counted and statistically analyzed (d , n = 3 individual experiments). All values are reported as the means ± S.E. ( error bars ), Student’s t -test; n.s, not significant ( p > 0.05); * p < 0.05; ** p < 0.01.

Article Snippet: The experiments were performed using the following antibodies: monoclonal antibodies (mAbs) against integrin α5 (610634) and integrin β4 (611233) were obtained from BD Biosciences; the mAbs against human integrin α5β1 (MAB1999) and integrin β4 (MAB2059) were from Millipore; the mouse pAbs to EGFR (Sc-120), integrin α3 (Sc-6592), or integrin α3 (Sc-32237) and the rabbit pAbs to integrin β4 (Sc-9090) or EGFR (Sc-03) were from Santa Cruz Biotechnology; rabbit mAbs to EGFR (catalog no. 4267), p-EGFR (catalog no. 3777), ERK1/2 (catalog no. 9102), p-ERK1/2 (catalog no. 4370), AKT (catalog no. 9272), and p-AKT (catalog no. 4060) were from Cell Signaling Technology; and, the mAb against α-tubulin was from Sigma.

Techniques: Stable Transfection, Knock-Out, Cell Culture

Journal: Scientific Reports

Article Title: N -Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

doi: 10.1038/srep33507

Figure Lengend Snippet: ( a ) The cell lysates from WT and unglycosylated mutant CHO-B2/EGFR cells (as an input; bottom panels ) were directly blotted with indicated antibodies or IP with anti-GFP agarose ( top panels ), E4-PHA-agarose or L4-PHA-agarose ( middle panels ), and then subjected to WB, reciprocally followed by E4-PHA and DSA lectins or anti-integrin α5 antibody for detection. ( b ) Comparison the effects of GnT-III on EGFR-α5 and EGFR-β4 complex formation in WT, S3–5, and S3–5, 11 HeLa cells. The doxycycline (DOX)-inducible GnT-III overexpression stable cell lines were established as described under the “Methods” section. The indicated cells were cultured in the presence (+) or absence (−) with 1 μg/ml doxycycline for 72 h, and the cell extracts were IP with anti-EGFR antibody ( top panels ) or anti-GFP agarose ( middle panels ), and then subjected to WB, reciprocally followed by anti-integrin α5, β4, or EGFR antibodies for detection. The whole cell extracts were also subjected to WB as an “input” using anti-α-tubulin antibody or E4-PHA and DSA lectins ( bottom panels ).

Article Snippet: The experiments were performed using the following antibodies: monoclonal antibodies (mAbs) against integrin α5 (610634) and integrin β4 (611233) were obtained from BD Biosciences; the mAbs against human integrin α5β1 (MAB1999) and integrin β4 (MAB2059) were from Millipore; the mouse pAbs to EGFR (Sc-120), integrin α3 (Sc-6592), or integrin α3 (Sc-32237) and the rabbit pAbs to integrin β4 (Sc-9090) or EGFR (Sc-03) were from Santa Cruz Biotechnology; rabbit mAbs to EGFR (catalog no. 4267), p-EGFR (catalog no. 3777), ERK1/2 (catalog no. 9102), p-ERK1/2 (catalog no. 4370), AKT (catalog no. 9272), and p-AKT (catalog no. 4060) were from Cell Signaling Technology; and, the mAb against α-tubulin was from Sigma.

Techniques: Mutagenesis, Over Expression, Stable Transfection, Cell Culture

Journal: Scientific Reports

Article Title: N -Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

doi: 10.1038/srep33507

Figure Lengend Snippet: The site-11 N -glycosylation of integrin α5, which contains abundant branching structures, can serve as a “switch”, which can turn on the EGFR-integrin α5β1 complex formation to restrict the EGFR internalization as described previously , resulting in an inhibition of EGFR-related cellular signaling for cell proliferation (left panel). Meanwhile, the N -glycosylation can also switch off the EGFR-integrin α6β4 complex formation due to a mutual competition as described above, which is well known to lead to an activation of EGFR and its downstream signaling (right panel).

Article Snippet: The experiments were performed using the following antibodies: monoclonal antibodies (mAbs) against integrin α5 (610634) and integrin β4 (611233) were obtained from BD Biosciences; the mAbs against human integrin α5β1 (MAB1999) and integrin β4 (MAB2059) were from Millipore; the mouse pAbs to EGFR (Sc-120), integrin α3 (Sc-6592), or integrin α3 (Sc-32237) and the rabbit pAbs to integrin β4 (Sc-9090) or EGFR (Sc-03) were from Santa Cruz Biotechnology; rabbit mAbs to EGFR (catalog no. 4267), p-EGFR (catalog no. 3777), ERK1/2 (catalog no. 9102), p-ERK1/2 (catalog no. 4370), AKT (catalog no. 9272), and p-AKT (catalog no. 4060) were from Cell Signaling Technology; and, the mAb against α-tubulin was from Sigma.

Techniques: Inhibition, Activation Assay