Cell culture regents were from Lonza (Walkersville, MD). Pharmacological inhibitors bafilomycin A1 (from Streptomyces griseus), Dynasore (3-Hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide), E-64D ((2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane Ethyl Ester) and leupeptin were from EMD Biosciences/Merck (San Diego, CA), Dyngo 4a, PitStop2 and PitStop2 negative control were from Ascent Scientific/Abcam (Princeton, NJ), whereas epoxomycin and clasto-Lactacystin β-Lactone were from Boston Scientific (Natick, MA).
Caveolin 1−/− (Cav1−/−) mice on C57Bl/6 background (strain B6.Cg-Cav1tm1Mls
were obtained from The Jackson Laboratory (Bar Harbor, Maine). The mice were maintained by heterozygous (Cav1+/−) cross breeding to generate to generate Cav1−/−, Cav1+/− and WT littermates. Every 5 generations Cav1−/− mice were bred to C57Bl/6J mice from Jackson Laboratory. Cavin-1−/− mice on C57Bl/6 background were from Dr. P. Pilch, Boston University 
Mouse polyoma virus middle T antigen immortalized mouse lung ECs (MLEC) obtained from Cav1KO (MLEC-Cav1KO), Cav1 ECRC (MLEC-Cav1 ECRC) and WT (MLEC-WT) mice, as described 
. The cells were flow sorted for surface expression of both CD31 and PV1 using a FACSAria sorter (BD Biosciences), as described below. All mouse lung ECs were cultured on plastic in MLEC growth medium consisting of endothelial growth medium 2 (EGM2) (Lonza, Walkersville, MD) supplemented with 15% heat inactivated fetal bovine serum (Hyclone), 100 µg/ml penicillin, 100 µg/ml streptomycin and 100 µg/ml glutamine (Invitrogen, Carlsbad, CA). HUVEC were obtained from Lonza and were cultured in EGM2 medium. The hybridoma secreting the rat anti-mouse PV1 IgG2a mAb MECA-32 
were from the Developmental Studies Hybridoma Bank, University of Iowa.
Rabbit anti caveolin 1 pAb (cat# 610060) and mouse anti –PTRF (cavin1) mAb, clone #4 (Cat# 611259) were purchased from BD Bioscience (San Diego, CA). Rat anti-mouse CD31 (PECAM-1) clone MEC13.3 – APC (Cat# 102510) was from BioLegend. Mouse anti-beta-actin mAb (AC40) was from Sigma (St.Louis, MO). Rabbit anti ERK1/2 mAb was from Cell Signaling (Beverly, MA). Goat anti-CD31 (PECAM-1) pAb (M-20, cat# sc-1506) and goat anti-Cdh5 (VE Cadherin, CD146) pAb (C-19, cat# sc-6458) were from Santa Cruz. The unlabeled and HRP-conjugated rabbit anti-chicken IgY and the goat anti-mouse IgG-HRP were from Biodesign (Kennebunk, ME). Rat anti-mouse PV1 IgG2a mAb, clone MECA-32 mAb was produced in serum free media by BioXCell, Lebanon, NH. Chicken anti-mouse PV1C pAb
was raised in chickens against the last 12 aa of mouse PV1 C terminus, as described in the past for chicken anti-human PV1C pAb 
Primary antibody labeling with fluorophores
Affinity purified primary antibodies rat anti-mouse PV-1 mAb clone MECA-32 and chicken anti-mouse PV-1C pAb, were labeled with either Alexa (488, 568 or 647) fluorophores (Molecular Probes, Invitrogen), as per manufacturer's instructions.
MLEC-Cav1KO, MLEC-Cav1 ECRC and MLEC-WT cells were cultured in 10 cm dishes to confluence. The cells were incubated (30 min, 10°C) live with 1
500 anti-CD31-APC (BioLegend) and anti-PV1-AF488 mAb clone MECA-32 (1 µg/ml) diluted in MLEC growth medium. The excess antibody was rinsed (3×5 ml) with sterile PBS (Invitrogen) and the cells dissociated nonenzymatically with cell dissociation solution (Sigma). The gating parameters were set on CD31+/PV1+ positive cells.
Specimen preparation for electron microscopy was done as before 
. Cav1−/− mice or WT littermates were perfused (10 min, RT) under anesthesia, with oxygenated DMEM through the left ventricle, followed by fixation by perfusion (10 min at RT) with 2.5% glutaraldehyde and 3% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.3). Specimens were taken from different tissues and trimmed into small blocks. The blocks were immersed into fresh fixative (1 h at RT), washed twice (15 min, RT) in 0.1 M cacodylate, postfixed in Palade's OsO4
(1 h on ice), en bloc-stained in Kellemberger's uranyl acetate (overnight at RT), dehydrated in graded ethanol, and embedded in LX112 resin (Ladd Research Industries, Burlington, VT). Thin sections (40 nm) with a Leica Ultracut (UC-6) using an ultrasonic oscillating diamond knife (Diatome, US), stained with lead citrate, and examined and photographed under an electron microscope (JEOL 1010).
Morphometry was done as before 
on lungs and kidneys obtained from Cav1−/− and WT mice. The measurements were obtained from capillaries found in 15–20 sections per animal per tissue (n
3 mice per group, 5 blocks per animal, 3–4 sections per block).
In the case of the lung, the number of caveolae per µm of EC length was determined by counting the number of uncoated plasmalemma invaginations in the 50–100 nm size range 
at the luminal or abluminal front of the ECs in capillaries (i.e.
blood vessels with <10 µm diameter) 
. The total membrane length examined was determined by summing the total length of luminal membrane to the total length of abluminal membrane. The data obtained in all animals per group was expressed as number of caveolae per µm endothelial membrane length. Student's t
test was used to determine statistical significance between WT and Cav1−/− groups.
In the case of the kidney, we have determined the number of caveolae, TEC and fenestrae per µm EC length in the ECs of the peritubullar capillaries following the methodology previously described by Millici, et al., 
. By this, caveolae, TEC and fenestrae were counted only in the areas in which EC thickness was less than 400 nm, as these are the areas where TEC and fenestrae occur. In the case of uncertainty as to whether a transendothelial opening was a TEC (i.e. with two diaphragms) or a fenestra (i.e. only one diaphragm) we have labeled this as an unknown (U). The data is expressed as the total number of structures found in 45–50 sections from each genotype divided by the membrane length. Because the fenestrae and TEC involve both fronts of the EC plasma membrane (i.e.
luminal and abluminal) we have considered the total membrane length as an average of the luminal and abluminal plasma membrane.
Colocalization of PV1 and Cav1 by Total Internal Reflection Fluorescence Microscopy (TIRFM)
MLEC-wt cells were seeded at 50% confluence on glass bottom dishes (MatTek) and were trasnfected with Cav1-EGFP 
, using Fugene 6 (Roche). Twenty four to forty eight hours post transfection the cells were labeled live with 1.5 µg/ml MECA-32-Alexa 568 mAb for 30 min at 4°C in MLEC growth medium, the cells rinsed (3×, RT) in MLEC growth medium and immediately used for live TIRFM.
TIRFM images were acquired live as before 
, using an Olympus IX71 inverted microscope equipped with a temperature-controlled stage set at 32°C, a 1.45 NA 60× TIRFM lens (Olympus), back-illuminated electron-multiplying charge-coupled device camera (512×512, 16-bit; iXon887; Andor Technologies), and controlled by Andor iQ software (Andor Technology). Excitation was achieved using a 488-nm and a 514-nm line of laser, and exposure times were 0.1–0.2 s and acquired at 0.5–4 Hz. The calculated evanescent field depth was 100 nm.
Due to optical characteristics of the two wavelengths leading to an uneven signal in the two wavelengths in TIRFM, the colocalization was done by scoring puncta positive for the two labels and not by the usual thresholding and calculation of the colocalization index but manually.
MLEC-WT were seeded at 70–90% confluence in 12 well plates and transfected with different DNA constructs using Superfect (Qiagen), as per manufacturer's instructions. The DNA constructs were as reported before 
: EGFP-clathrin light chain from J. Keen (Thomas Jefferson University, Philadelphia, PA) and dynamin 2 wt-EGFP and dynamin 2(K44A)-EGFP in pEGFP-N1 vector from M. McNiven 
(Mayo Clinic, Rochester MN). pEGFP-N1 empty vector was from Clontech. Forty-eight hours post transfection the cells were labeled with fluorescent anti-PV1 and processed for either confocal microscopy or flow cytometry.
MLEC were serum starved (2 h, 37°C, EBM2), labeled (30 min, 10°C, EBM2+1%BSA) with fluorescent anti-PV1 (5 µg/ml) and rinsed (3×, RT) in PBS containing calcium and magnesium (PBS-CM) and chased (37°C, MLEC growth medium) for different amounts of time. After 0, 15, 60 and 120 min the cells were rinsed (2×30 sec, RT) at low pH to facilitate detachment of non-internalized antibodies, rinsed 1× in neutral PBS, fixed (10 min, RT) in 4% paraformaldehyde in PBS-CM, rinsed again in PBS-CM containing DAPI, mounted in PermaFluor (Thermo Fisher) mounting medium and examined by confocal fluorescence microscopy using a Zeiss 510 Meta confocal system equipped with a 63× oil immersion objective and appropriate lasers. Stacks of images were acquired with the pinhole set at 1 Airy unit and processed using ImageJ software. For PV1 internalization, stacks were transformed through the maximum intensity projection function to obtain global images of the cells. Figures were prepared using Adobe Photoshop and Adobe Illustrator CS3 software.
Isolation of total membranes from lungs and kidneys
Lung and kidney membrane lysates were obtained from WT, Cav1−/−
mice, as described in the past 
. The mice were anesthetized with a mixture of ketamine : xylazine : acepromazine (3
0.25). The lungs and the kidneys were immediately flushed free of blood by perfusion (10 min, 25°C) with oxygenated phenol-red free HBSS, via the pulmonary artery or the left ventricle, respectively. The organs were freshly collected, weighed, minced and homogenized (20 strokes, Teflon pestle-glass Thomas type BB homogenizer) in an ice-cold buffer (1
v) containing 25 mM Hepes, pH 7.2, 250 mM sucrose, 2 mM MgCl2
and a protease inhibitors cocktail (10 µg/ml each leupeptin, pepstatin, o-phenantrolin, E-64 and 1 mM PMSF). The homogenate was filtered through 53 µm nylon net and centrifuged for 15 min at 500×g to yield a nuclei/cell debris pellet and a postnuclear supernatant (PNS). The PNS was further fractionated by centrifugation (1 h, 4°C, 100,000×g, using a TLA45 rotor) in a total membranes pellet and a cytosolic supernatant. The membrane pellet was solubilized in 200 µl 10 mM Tris, pH 6.8, 0.5%SDS, and protease inhibitors (Sigma). Protein concentration was determined by a bicinchoninic acid method (Pierce, Rockford, IL/USA) using BSA standards standards prepared in solubilization buffer, as described previously 
. Equal amounts of protein (20 µg) were adjusted to 1× reducing SDS-PAGE sample buffer, boiled for 5 min, resolved by 12% or 8% SDS-PAGE, transferred to PVDF membrane and probed by immunoblotting. Antibodies used were either the rat anti-mouse PV1 MECA-32 mAb, the chicken anti-mouse PV1C pAb described here, rabbit anti caveolin 1 pAb, mouse anti-Cavin-1/PTRF, goat anti CD31 pAb, goat anti-VE Cadherin pAb and rabbit anti-Cavin 2/SDPR (Abcam). The lung membrane lysates were obtained from WT, Cav1KO, Cav1+/−
and Cavin-1−/− mice.
CD31 and PV1 positive MLEC-WT, -Cav1KO or -Cav1-ECRC grown to confluence in 2×15 cm dishes were scrapped and collected by centrifugation in tubes, the pellets resuspended in 1 ml ice-cold buffer (containing 25 mM Hepes, pH 7.2, 250 mM sucrose, 2 mM MgCl2 and a broad spectrum protease inhibitors cocktail - Sigma), homogenized (4°C, 20 strokes, Teflon pestle-glass Thomas type A homogenizer) followed by sonication (3×30 sec bursts) using a Branson sonicator. The homogenate was fractionated as described above for the lung and kidney membranes.
MLEC-wt and MLEC-Cav1KO cells were grown to confluence in quadruplicate in 15 cm Petri dishes. Freshly harvested lungs and kidneys from WT, Cav1−/− and cavin-1−/− mice were used for total RNA isolation. Total RNA was isolated using Trizol (Invitrogen), as per manufacturer's instructions.
Real-time quantitative PCR
RNA integrity and quality were determined using Bioanalyzer (Agilent) and NanoDrop (Thermo-Fisher). One microgram of total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA amplified from 10 ng RNA were used in triplicate for quantitative real-time PCR using Taqman® Gene Expression Assays (ABI) designed for mouse PV1 (PV1/Plvap), Cav1, cavin-1 and Actin B (ActB) mRNA detection and the TaqMan® Gene Expression Master Mix, as per manufacturer's instructions. The PCR was performed on ABI 7500 Real Time PCR System with SDS software. The comparative CT method (2−ΔΔCT) of relative quantitation was used to compare the two genotypes.
PNGase treatment to remove N-linked glycans
Solubilized membrane (Mem) and cytosolic (Cyt) proteins (100 µg) were treated with PNGase F (New England Biological), as per manufacturer's instructions. Controls were incubated in the same conditions except that PNGase F was omitted. The samples were resolved by 8% SDS-PAGe and the proteins transferred to PVDF membrane and immunoblotted with chicken anti-PV1C pAb.
35S metabolic labeling of MLEC
MLEC-wt and MLEC-Cav1KO cells were grown to confluence in 60 mm dishes. Cells were Met and Cys starved by incubation (2 h, 37°C) in 35
S labeling medium consisting of EBM medium lacking these Met and Cys (Lonza). 100 µCi of 35
S Translabel (Perkin-Elmer) consisting of a mix of 35
S labeled Met and Cys were added to the 35S labeling medium and the cells were incubated for 10 min at 37°C to allow for the 35
S-labeled aminoacids to be incorporated in proteins during translation. Mouse PV1 has 8 methionines and 10 cysteines in its primary sequence. After 2 washes in EBM2, the cells were chased for 0, 5 min, 15 min, 30 min or 1, 2, 4, 8, 12 and 24 h when the cells were rinsed in PBS, collected by scrapping in 1 ml solubilization buffer [1% Triton X-100 in 10 mM Tris-Cl, pH 7.4, 150 mM NaCl and protease inhibitors cocktail (Sigma cat# P8340)] followed by incubation (4°C, 2 h) with end over end rotation to complete the solubilization, These conditions are known to efficiently solubilize PV1 
. The samples were centrifuged (1 h, 4°C, 100,000×g) to remove the insoluble material as a pellet. The supernatant, containing the solubilized PV1, was added to 50 µl (settled gel) of chicken anti-mouse PV1 directly coupled to AffiGel 10 beads, as described before 
. Beads coupled with preimmune chicken IgG were used as controls. The samples were incubated (o/n, 4°C) by rotation after which the beads were collected by centrifugation (5 min, 4°C, 300×g) and washed 3 times at 4°C with solubilization buffer. PV1 bound to the beads was solubilized in SDS-PAGE sample buffer, resolved by 8% SDS-PAGE, the gel treated with Amplify (GE Healthcare), vacuum dried and exposed to a multipurpose standard (MS) phosphor storage screen (Kodak). The signal was imaged using a Typhoon 9400 scanner (Molecular Dynamics, GE Healthcare) and quantified using ImageJ or GelEval v1.35 (FrogDance, UK) software. Data from three separate experiments were used to obtain the degradation curves.
Labeled cells were analyzed by either using a FacsCalibur or a CANTO flow cytometer controlled by either CellQuest or DIVA software, respectively (BD Biosciences). The data analysis was carried out using FlowJo (Tree Star, Ashland, OR) software. Each experiment had 4–8 samples per time point and was repeated at least three times. Median fluorescence from at least 10,000 live cells was calculated in each sample. Statistical significance was calculated using Student's t test.
Evaluation of cell surface PV1 levels by flow cytometry
Either MLEC-wt or MLEC-cav1KO cells were labeled live and while adherent with 1.5 µg/ml MECA-32-Alexa 647 mAb for 30 min at 4°C in MLEC growth medium. The cells were rinsed (3×, RT) in PBS and non-enzymatically detached using EDTA (Cell Dissociation Solution, Sigma). The cells were mixed with an equal volume of 1% BSA in PBS, and kept on ice in the dark until examined by flow cytometry.
Evaluation of PV1 internalization rate by flow cytometry
Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 µg/ml) in EBM2 supplemented with 2% BSA. After washing (3×, EBM2) the excess primary antibody off, the cells were incubated with full MLEC growth medium at 37°C for the indicated periods of time to allow for the internalization of the antibody. To determine the internalized fraction, the cells were washed once (30 s, RT) in acidic PBS, pH 2.5, once in neutral PBS and detached by incubation (10 min, 37°C) in a mixture of trypsin/EDTA (Lonza). The combination of acid wash and the trypsin treatment were very effective in removing the surface anti-PV1, demonstrated on separate control samples incubated at 4°C.
To determine the initial surface pool of PV1, cells were incubated with fluorescent anti-PV1 as above, rinsed in neutral PBS and non-enzymatically detached using EDTA (Cell Dissociation Solution, Sigma). These conditions do not disrupt the anti-PV1 - PV1 interaction on cell surface.
The cell suspensions were mixed with an equal volume of 1% BSA in PBS, and kept on ice in the dark until examined by flow cytometry. The average median fluorescence was calculated from each time point and the percentage of internalized PV1 was calculated from the ratio of internalized/initial anti-PV1 signal. Fluorescent rat IgG2a was used as isotype control for MECA-32 antibody.
Evaluation of PV1 degradation pathway
Equal numbers of MLEC-WT and MLEC-Cav1KO were seeded into 6 cm dishes at 90% confluence the evening before and cultured in full growth medium until the next day when the medium was replaced with MLEC growth medium containing either proteasome inhibitors (i.e.
epoxomycin or clasto-Lactacystin beta Lactone), lysosome inhibitors (i.e.
leupeptin, E64-D or bafilomycin A1) or DMSO vehicle. The cells were further incubated for 4 h, 8 h or 24 h at 37°C in a cell culture incubator with 5%CO2 atmosphere. The final inhibitor concentrations obtained from 1000-fold concentrated stocks in DMSO were as follows: 2 µM epoxomycin, 10 µM clasto
-Lactacystin β-Lactone, 10 µM E-64D, 50 µM leupeptin, 1 µM or 10 µM bafilomycin A1. At the end of the experiment cells were rinsed (2×, RT) in 5 ml PBS and then solubilized (1 h, 4°C) in .5 ml RIPA buffer (1% Triton X-100, 0.4% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl in 25 mM Tris, pH7.6) with protease inhibitors. The samples were centrifuged (1 h, 4°C, 100,000×g) to remove the insoluble material as a pellet. The supernatant was transferred to a fresh tube and used to determine the protein concentration using the BCA assay and standards made in RIPA buffer. Equal amounts (20 µg) of proteins from different samples were resolved by 8% SDS-PAGE, transferred to PVDF membranes and immunoblotted with either rat-anti-mouse PV1 mAb MECA-32 or chicken anti-mouse PV1C pAb, as described 
Evaluation of the effect of dynamin and clathrin inhibitors on PV1 internalization
To determine the effects of clathrin mediated uptake inhibitor PitStop2 
or the dynamin inhibitors Dynasore (10 and 80 µM) 
or Dyngo4a (30 µM) 
on PV1 internalization, cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with Alexa647-MECA-32 mAb (1.5 µg/ml) in EBM2 supplemented with either: 25 µM PitStop2 (Ascent Scientific), 25 µM PitStop2 negative control compound (Ascent Scientific), 10 µM or 80 µM dynasore (EMD Chemicals) or 30 µM Dyngo4a (Ascent Scientific). Negative controls consisted of cells not treated or treated with DMSO vehicle (1 µl/ml medium). After washing (3×, EBM2) the excess primary antibody off, the cells were incubated (37°C, 15 min or 60 min) with either EMB2 or full MLEC growth medium supplemented with inhibitors or vehicle. For each inhibitor we have determined the total
surface PV1 signal at t0 min, t15 min and t60 min by nonenzymatic digestion as well as the internalized fraction
using the combination of acid wash and trypsin/EDTA, as described above. For each inhibitor the samples were run in quadruplicate in three separate experiments. Because protein in the medium might inactivate the inhibitors, for each inhibitor the assay was carried out in absence and presence of protein in the medium (EBM2 vs. full growth MLEC medium).
Positive controls for each inhibitor effectiveness consisted of Alexa647 labeled human transferrin or EGF (Invitrogen, Molecular Probes). Serum starved (o/n, 37°C, EBM2+1%BSA) endothelial cells were incubated (30 min, 10°C) in serum-free medium supplemented with inhibitors and either 25 µg/ml transferrin-F647 or 1 µg/ml biotinEGF-streptavidin-Alexa647, the excess label washed away, the cells incubated (10 min, 37°C) in growth medium with inhibitors to allow internalization of the surface bound label, when the cells were acid washed, resuspended using trypsin/EDTA and examined by flow cytometry.