Journal:

Article Title: The light-activated signaling pathway in SCN-projecting rat retinal ganglion cells

doi: 10.1111/j.1460-9568.2006.04777.x

Figure Lengend Snippet: Ion channel expression in melanopsin-containing RGCs. (A) CNG channels do not colocalize with melanopsin. Retinal sections were immunostained for melanopsin (green) and CNGA1 (top, red), CNGA2 (middle, red) and CNGA3 (bottom, red). DAPI-staining of cell nuclei (blue) was used to visualize retinal layers. (B) TRPC6 channels are found in most RGCs, including those that express melanopsin. Melanopsin (green) was visualized by tyramide signal amplification, and TRPC6 (red) was visualized using conventional immunostaining techniques (see Methods). Scale bars, 20 μM. Abbreviations: POS (photoreceptor outer segments); ONL (outer nuclear layer); OPL (outer plexiform layer); INL (inner nuclear layer); IPL (inner plexiform layer); GCL (ganglion cell layer).

Article Snippet: Monoclonal antibodies CRO2H7 against CNGA2 from olfactory receptor neurons, and CNC9C1 against CNGA3 from cone photoreceptors, were gifts of Dr U. Benjamin Kaupp [Forschungszentrum, Julich, Germany; ( Meyer et al. , 2000 )].

Techniques: Expressing, Staining, Amplification, Immunostaining

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: VE-cadherin endocytosis is mediated through a clathrin-, AP-2–, and dynamin-dependent pathway. (A and B) Cell surface IL-2R-VE-cadcyto was labeled with IL-2R antibodies at 4°C. Cells were incubated at 37°C for 5 min, washed in a low pH buffer at 4°C to remove surface bound antibody, and then processed for immunofluorescence to visualize intracellular IL-2R-VE-cadcyto (A, a′, c′, and e′). Antibodies against the myc epitope tag at the carboxyl terminal domain of the IL-2R-VE-cadcyto were used to verify expression of the polypeptide (A, b′, d′, and f′). Note that untreated cells exhibited high levels of internalized IL-2R-VE-cadcyto, whereas treatment with K+ depletion or chlorpromazine completely inhibited internalization. (C–F) MECs were infected with adenoviruses expressing either GFP (C, a′ and b′) or dominant negative DynII (C, c′ and d′). The DynII completely blocked internalization of Alexa Fluor 555–conjugated transferrin (C and D). To monitor internalization of VE-cadherin, MECs were either untreated (E, a′ and b′) or treated with 100 μM chloroquine for 3 h (E, c′–f′). Note that intracellular vesicular accumulation of VE-cadherin is reduced in cells expressing DynII (E, e′ and f′, and F). (G–K) HMEC-1 cells were transfected with an siRNA oligo targeted to the AP-2 μ subunit or a nontargeting control siRNA. Lysates prepared from cells treated with control siRNA or AP-2 siRNA were separated by SDS-PAGE and immunoblotted for AP-2 to confirm AP-2 knockdown (G). Western blot and immunofluorescence reveals ∼75% reduction in AP-2 levels in siRNA-treated cells compared with control, as well as an 85% decrease in transferrin internalization (G–I). IL-2R-VE-cadcyto internalization assays were performed and AP-2 siRNA-treated cells (J, c′ and d′) exhibited an 80% decrease in internalization of IL-2R-VE-cadcyto compared with control (J, a′ and b′), indicating a requirement for AP-2 in VE-cadherin internalization (K). Error bars, SEM; n > 20 cells. Scale bar, 20 μm.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Labeling, Incubation, Immunofluorescence, Expressing, Infection, Dominant Negative Mutation, Transfection, SDS Page, Western Blot

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: The VE-cadherin cytoplasmic tail specifically associates with clathrin and AP-2. MECs expressing IL-2R or IL-2R-VE-cadcyto were treated with DSP (lanes 2, 4, 7–8, and 11–12) or DMSO vehicle (lanes 1, 3, 5–6, 9–10, and 13–14), and extracted in detergent, and proteins were immunoprecipitated with beads alone (lanes 5–8), beads coated with AP-2 α antibodies (lanes 8–12), or beads coated with clathrin antibodies (CHC; lanes 13–14). Western blot analysis using antibodies against IL-2R to detect IL-2R or IL-2R-VE-cadcyto demonstrates that the IL2R-VE-cadcyto specifically interacts with AP-2 (lane 12) and clathrin (lane 14). Inputs represent 5% of sample.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: The VE-cadherin cytoplasmic tail specifically colocalizes with clathrin and AP-2. MECs expressing IL-2R, IL-2R-VE-cadcyto, or IL-2R-Dsg3cyto were labeled for 30 min at 4°C with IL-2R antibodies, transferred to 37°C for 5 min, and then processed for immunofluorescence microscopy. As a positive control, internalization of transferrin receptor was monitored by labeling MECs with fluorescently conjugated transferrin at 4°C, transferring cells to 37°C for 5 min, and then processing for immunofluorescence microscopy. (A and B) Colocalization of transferrin, IL-2R, IL-2R-VE-cadcyto, or IL-2R-Dsg3cyto with clathrin or caveolin was monitored. Colocalization was quantified as the percentage of transferrin, IL-2R, IL-2R-VE-cadcyto, or IL-2R-Dsg3cyto that colocalize with clathrin or caveolin, using Metamorph software. (C and D) Colocalization of IL-2R and IL-2R-VE-cadcyto with AP-2 was also measured. Colocalization was quantified as the percentage of transferrin, IL-2R, or IL-2R-VE-cadcyto that colocalize with AP-2. Error bars, SEM; n = 25 cells. Scale bar, 20 μm.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Expressing, Labeling, Immunofluorescence, Microscopy, Positive Control, Transferring, Software

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: Exogenous expression of p120 prevents IL-2R-VE-cadcyto from colocalizing with clathrin and AP-2 in a manner dependent on the interaction of p120 with the VE-cadherin JMD. (A) p120 was coexpressed in MECs with either IL-2R-VE-cadcyto or IL-2R-VE-cadJMD-AAA, which is unable to bind to p120, and internalization assays were conducted as described in Figure 3. Cells were fixed and processed for triple-label immunofluorescence to monitor the IL-2R-VE-cadherin polypeptides, p120, and clathrin. (B) Colocalization was quantified as the percentage of IL-2R-VE-cadcyto or IL-2R-VE-cadJMD-AAA that colocalize with clathrin. In the presence of p120, colocalization between IL-2R-VE-cadcyto and clathrin is greatly reduced. High levels of colocalization between IL-2R-VE-cadJMD-AAA and clathrin in cells expressing exogenous p120 demonstrate that p120 must bind to the VE-cadherin JMD to prevent VE-cadherin recruitment into clathrin-enriched membrane domains. (C) Colocalization between IL-2R-VE-cadcyto and AP-2 was also monitored. In the presence of p120, IL-2R-VE-cadcyto colocalization with AP-2 is dramatically reduced (D). Error bars, SEM; n = 25 cells. Scale bar, 20 μm.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Expressing, Immunofluorescence

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: A p120 Rho-uncoupled mutant prevents entry of VE-cadherin into membrane compartments containing clathrin and AP-2. (A and C) MECs were infected with adenovirus carrying IL-2R-VE-cadcyto and either p120 1A or p120 4A K622,628A. Cells were then processed for immunofluorescence to determine the extent of colocalization between IL-2R-VE-cadcyto and clathrin (A) or AP-2 (C) in cells expressing either wild-type or Rho-uncoupled p120 (p120 4A K622,628A). Colocalization was quantified as the percentage of IL-2R-VE-cadcyto that colocalize with clathrin or AP-2. The RhoA-uncoupled p120 mutant was as effective at preventing colocalization of IL-2R-VE-cadcyto as wild-type p120 (B and D). Error bars, SEM; n = 25 cells. Scale bar, 20 μm.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Mutagenesis, Infection, Immunofluorescence, Expressing