task3  (Alomone Labs)

Journal: Frontiers in Cellular Neuroscience

Article Title: Synaptic Scaffolds, Ion Channels and Polyamines in Mouse Photoreceptor Synapses: Anatomy of a Signaling Complex

doi: 10.3389/fncel.2021.667046

Figure Lengend Snippet: Localization of inward rectifier potassium channel Kir2.1 in mouse retina. (A) Immunostaining for Kir2.1 (rabbit antibody; red) with DAPI counterstaining (blue) and PSD95 (green) to label photoreceptor terminals. Labels for retinal nuclear layers are as in Figure 1 . Kir2.1 is diffusely present in the IPL, but strongly labeled at the inner limiting membrane (arrowheads) and in the OPL. Five μm confocal stack. (B) Higher magnification view of the OPL reveals Kir2.1 (red) labeling largely below the photoreceptor terminals (green; PSD95 labeling), but with some diffuse and punctate labeling among the terminals. Two μm confocal stack. (Ci) Kir2.1 labeling (guinea pig antibody; red) colocalizes with Calbindin labeled horizontal cells (green) in the OPL. (Cii) Kir2.1 labeling in isolation. (Di) Another view of horizontal cells in the OPL (Calbindin labeling; green) along with mGluR6 labeling (magenta) to show locations of On bipolar cell dendritic tips. Clusters of mGluR6 label indicate cone terminals; three examples are indicated with paired arrowheads. Six μm confocal stack. (Dii) Kir2.1 labeling (rabbit antibody; red) in the same section. (Diii) Merged view of all three labels. Kir2.1 shows both diffuse and punctate labeling in the vicinity of horizontal cell axon terminal projections contacting rods, but little labeling near clusters of dendritic processes contacting cones. (Ei) Higher magnification view of a horizontal cell labeled with Calbindin antibody (green). Several representative axon terminal tips are highlighted with arrowheads. One μm confocal stack. (Eii) Kir2.1 labeling in the same section. (Eiii) Merged view of the two labels shows that tips of horizontal cell axon terminal processes contain punctate clusters of Kir2.1 labeling (arrowheads). (F) Average mRNA expression levels of Kir2.1 (Kcnj2) and kainate receptor subunits GluR6 (Grik2) and GluR7 (Grik3) in mouse retina from single-cell transcriptome data. Kir2.1 is most prominently expressed in horizontal cells and essentially absent from Müller glia. The kainate receptor subunit GluR6 is also found in horizontal cells and Off bipolar cells, but GluR7 is absent from these cell types. Data adapted from Hoang et al. (2020) .

Article Snippet: Antibodies used included rabbit anti-SAP-102 (Thermo Fisher Scientific, Waltham, MA; AB_2546592 ), goat anti-SAP-102 (Abcam, Cambridge, MA; AB_777828 ), mouse anti-SAP-97 clone K64/15 (UC Davis/NIH NeuroMab Facility, Davis, CA; AB_2091920 ), mouse IgG1 anti-Chapsyn-110 clone N18/30 (UC Davis/NIH NeuroMab Facility; AB_2277296 ), mouse IgG2a anti-PSD-95 clone K28/43 (UC Davis/NIH NeuroMab Facility; AB_444362 ), mouse anti-CASK (Antibodies-online, Inc., Limerick, PA), sheep anti-mGluR6 (gift of Dr. Catherine Morgans; ), mouse anti-GluR6/7 clone NL9 (Millipore-Sigma, Burlington, MA; Cat# 04-921, AB_1587072 ), rabbit anti-Kir2.1 (Alomone, Jerusalem, Israel; AB_2040107 ), guinea pig anti-Kir2.1 (Alomone; AB_2340970 ), rabbit anti-Connexin 57 (Invitrogen, Camarillo, CA; AB_2314266 ), rabbit anti-Pannexin 1 (Alomone; AB_2340917 ), rabbit anti-Pannexin 2 (Thermo Fisher Scioentific; AB_2533518 ), rabbit anti-Calbindin (Swant, Bellinzona, Switzerland; AB_1000034 ), mouse anti-Calbindin (Abcam; AB_1658451 ), mouse anti-Glutamine Synthetase (Millipore-Sigma; AB_2110656 ), and rabbit anti-SLC18B1 (Sigma Life Sciences, St. Louis, MO; AB_10600797 ).

Techniques: Immunostaining, Labeling, Isolation, Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

doi: 10.1074/mcp.RA120.002071

Figure Lengend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

Article Snippet: Immunofluorescence (IF) Staining ExperimentsIF staining experiments were performed utilizing either HEK293 cells or freshly isolated rat ventricular myocytes, as previously described ( ) using the following primary and secondary antibodies: PKP4 (ThermoFisher Scientific, PA5-66855, dilution 1/500), Kir2.1 (Alomone Labs, Jerusalem, Israel, AGP-044, dilution 1/20), Actinin (Sigma, SAB4503474, 1/300), GM130 (Novus Biologicals, NBP2-53420; dilution 1/200), FLAG (Sigma-Aldrich; F3165, dilution: 1/50), Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-guinea pig and Alexa Fluor 647 donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA, dilution 1/400).

Techniques: Transformation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

doi: 10.1074/mcp.RA120.002071

Figure Lengend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

Article Snippet: Immunofluorescence (IF) Staining ExperimentsIF staining experiments were performed utilizing either HEK293 cells or freshly isolated rat ventricular myocytes, as previously described ( ) using the following primary and secondary antibodies: PKP4 (ThermoFisher Scientific, PA5-66855, dilution 1/500), Kir2.1 (Alomone Labs, Jerusalem, Israel, AGP-044, dilution 1/20), Actinin (Sigma, SAB4503474, 1/300), GM130 (Novus Biologicals, NBP2-53420; dilution 1/200), FLAG (Sigma-Aldrich; F3165, dilution: 1/50), Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-guinea pig and Alexa Fluor 647 donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA, dilution 1/400).

Techniques: Over Expression, CRISPR

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

doi: 10.1074/mcp.RA120.002071

Figure Lengend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

Article Snippet: Immunofluorescence (IF) Staining ExperimentsIF staining experiments were performed utilizing either HEK293 cells or freshly isolated rat ventricular myocytes, as previously described ( ) using the following primary and secondary antibodies: PKP4 (ThermoFisher Scientific, PA5-66855, dilution 1/500), Kir2.1 (Alomone Labs, Jerusalem, Israel, AGP-044, dilution 1/20), Actinin (Sigma, SAB4503474, 1/300), GM130 (Novus Biologicals, NBP2-53420; dilution 1/200), FLAG (Sigma-Aldrich; F3165, dilution: 1/50), Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-guinea pig and Alexa Fluor 647 donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA, dilution 1/400).

Techniques: Immunofluorescence, Staining, Isolation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

doi: 10.1074/mcp.RA120.002071

Figure Lengend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

Article Snippet: Immunofluorescence (IF) Staining ExperimentsIF staining experiments were performed utilizing either HEK293 cells or freshly isolated rat ventricular myocytes, as previously described ( ) using the following primary and secondary antibodies: PKP4 (ThermoFisher Scientific, PA5-66855, dilution 1/500), Kir2.1 (Alomone Labs, Jerusalem, Israel, AGP-044, dilution 1/20), Actinin (Sigma, SAB4503474, 1/300), GM130 (Novus Biologicals, NBP2-53420; dilution 1/200), FLAG (Sigma-Aldrich; F3165, dilution: 1/50), Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-guinea pig and Alexa Fluor 647 donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA, dilution 1/400).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

doi: 10.1074/mcp.RA120.002071

Figure Lengend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

Article Snippet: Immunofluorescence (IF) Staining ExperimentsIF staining experiments were performed utilizing either HEK293 cells or freshly isolated rat ventricular myocytes, as previously described ( ) using the following primary and secondary antibodies: PKP4 (ThermoFisher Scientific, PA5-66855, dilution 1/500), Kir2.1 (Alomone Labs, Jerusalem, Israel, AGP-044, dilution 1/20), Actinin (Sigma, SAB4503474, 1/300), GM130 (Novus Biologicals, NBP2-53420; dilution 1/200), FLAG (Sigma-Aldrich; F3165, dilution: 1/50), Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-guinea pig and Alexa Fluor 647 donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA, dilution 1/400).

Techniques: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography