Anti ve cadherin antibody

Cells seeded on 35-mm glass-bottomed dishes were fixed with 4% paraformaldehyde for 10 min and blocked in 2% bovine serum albumin for 1 h. Early endosomes were stained with anti-EEA1 antibody (BD Transduction, #610457) and Alexa Fluor 568–conjugated secondary antibodies (Thermo Fisher, #A-11004), S1PR1 was stained with anti-S1PR1 antibody (Cell Signaling Technology, #63335) and Alexa Fluor 488–conjugated secondary antibodies (Thermo Fisher, #A28175), VE-cadherin was stained with anti-VE-cadherin antibody (Santa Cruz Biotechnology, #sc-9989) and Alexa Fluor 488–conjugated secondary antibodies, F-actin was stained with Alexa Fluor 594 phalloidin (Thermo Fisher, #A12381), and nuclei were stained with DAPI. Confocal fluorescence microscopy was performed as mentioned above.

Heart tissues were collected after the mice were killed, fixed in 4% paraformaldehyde, embedded in tissue processing medium (O.C.T.), and cut horizontally at a 10-mm thickness. The slides were stained with specific anti–VE-cadherin antibody (1:200 dilution, sc-9,989; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for overnight. Human umbilical vein endothelial cells obtained from in vitro cell culture were fixed with 3.7% formaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 20 min, followed by blocking with 3% bovine serum albumin for 30 min at room temperature. Subsequently, cells were incubated with anti–VE-cadherin antibody (1:200 dilution, sc-9,989; Santa Cruz Biotechnology) at 4°C for overnight. Alexa Fluor 594 secondary antibody (1:500 dilution in PBS, A-11005; Invitrogen) was added to the section to visualize the staining. DAPI (blue) was used to counterstain the nuclei. The stained sections and cells were measured using Confocal Microscope (Leica Camera, Wetzlar, Germany).

Western blot was performed as described previously (7 (link)). Briefly, cellular proteins were prepared in ice-cold RIPA buffer containing protease inhibitor and quantified with Pierce BCA protein assay kit (23,225; ThermoFisher, Waltham, MA). Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose blotting membranes (10,600,003; GE Healthcare, Chicago, IL). The membranes were incubated with appropriate primary antibodies (1:1,000 dilution in 5% bovine serum albumin) followed by the peroxidase-conjugated secondary antibody (1:5,000 dilution in 5% nonfat milk). The signals were quantified using G:Box gel imaging system by Syngene (Bangalore, India). The following primary antibodies were used in the present study: anti–VE-cadherin antibody (1:200 dilution, sc-9,989; Santa Cruz Biotechnology), anti-HSPA12B antibody (1:1,000 dilution, a gift from Dr. Zhihua Han [ETSU, Johnson City, TN]), anti-claudin 5 antibody (1:1,000 dilution, ab131259; Abcam, Cambridge, United Kingdom), anti–ZO-1 antibody (1:1,000 dilution, 13,663 s; Cell Signaling Technology, Danvers, MA), anti–β-actin antibody (1:1,000 dilution; 3,700 s; Cell Signaling Technology), and anti-GAPDH antibody (1:1,000 dilution, Cell Signaling Technology, 2,118 s).

Cells were seeded on chamber slides, grown until semi-confluence and processed as previously described [53 (link)]. In brief, cells were fixed in 3.7% paraformaldehyde for 10 min at room temperature with or without a subsequent permeabilization step with 0.1% Triton-X in PBS for 20 min. The cells were blocked in 10% 2nd antibody host serum with 4% BSA in PBS for 1 h at room temperature, followed by overnight incubation at 4 °C with SUCNR1 antibody (Novus Biologicals, Littleton, CO, USA). The cells were incubated with conjugated 2nd antibody (Invitrogen) for 2 h at room temperature after a washing step. For cell membrane localization, conjugated wheat germ agglutinin (WGA, ThermoFisher Scientific) or anti-VE-cadherin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used. For subcellular receptor localization, we used CellLight Mitochondria-RFP (Invitrogen) to identify the mitochondria. The slides were mounted in DAPI-containing mounting medium and examined with confocal microscopy, z-stacks were acquired and images were processed with Fiji.