A21447

The antibodies were as follows: PI(3,4,5)P: anti‐PtdIns(3,4,5)P₃ monoclonal antibody (Z‐P345b, 1:200, Echelon Biosciences), GFP (rabbit, 1:500 Abcam ab290), phospho‐S6 ribosomal protein (Ser235/236) (91B2) (rabbit, 1:200, Cell Signaling 4857S), TUJ1 (βIII Tubulin) (mouse, 1:400, Promega G7121), PTEN (D4.3) XP (rabbit, 1:100, Cell Signaling 9188S), p110δ (rabbit, 1:500, Abcam ab1678), Brn3a (C‐20) (goat, 1:200, Santa Cruz sc‐31984). ARF6, (rabbit, 1:100, Abcam ab77581), GST (1:400, Abcam ab19256), anti‐goat Alexa Fluor 647 (1:1,000, A21447, Life Technologies), anti‐rabbit IgG‐conjugated Alexa Fluor 568 (A10042, 1:1,000, Thermo Fisher Scientific), anti‐mouse IgG‐conjugated Alexa Fluor 568 (A11004, 1:1,000, Thermo Fisher Scientific), anti‐rabbit IgG‐conjugated Alexa Fluor 488 (A‐21206, 1:1,000, Thermo Fisher Scientific) and anti‐mouse IgG‐conjugated Alexa Fluor 488 (A‐21202, 1:1,000, Thermo Fisher Scientific).

Confluent HUVECs seeded on 24-well plates were subjected to the scratch-wound assay and then processed for PLA using the Duolink In Situ Red Mouse/Rabbit Starter Kit (DUO92101-1KT, Sigma-Aldrich) as described by the manufacturer’s protocol. To probe interactions between vinculin and VE-cadherin, cells were incubated with an anti-vinculin antibody raised in rabbit (V4139, Sigma-Aldrich) and an anti-VE-cadherin antibody raised in mouse (sc-9989, Santa Cruz Biotechnologies). In parallel, cells were also incubated with an anti-VE-cadherin antibody raised in goat (AF938, R and D Systems) and subsequently with an anti-Goat fluorescent-conjugated secondary antibody (A21447, Thermo Fisher Scientific) to label adherens junctions. To quantify co-localization of PLA signal at adherens junctions, high-resolution Z-stack images at multiple positions on the wound edge were acquired on a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with the Zen black software with a Plan Apochromat 63x NA 1.40 oil DIC M27 objective. Briefly, PLA dots were quantified only at adherens junctions, using a similar approach to the co-localization studies described in the section ‘Immunostaining co-localization analysis’, using the VE-cadherin immunofluorescence staining to detect overlapping pixels between junctions and PLA signals.

Primary antibodies used in this study were: DDX25 (sc-51271) and Hiwi (sc-22685) from Santa Cruz Biotechnology; DDX4 (ab13840), EIF4A3 (ab32485), and SMG6 (ab87539) from Abcam; PIWIL1 (G82) from Cell Signaling Technology; FYCO1 (HPA035526 and SAB1400697) from Sigma-Aldrich; GAPDH (5G4) from HyTest; γH2AX antibody (05-636) and PIWIL2 clone 13E-3 (MABE363) from Millipore; SMG7 (A302-170A) from Bethyl Laboratories and hVasa (AF2030-SP) from R&D systems. Secondary antibodies conjugated with Alexa Fluor 488, 546, and 647 made in donkey were from Thermo Fisher Scientific (A-21202, A-21206, A-11055, A10036, A10040, A-11056, A-21203, A-21207, A-11058, A-31571, A-31573, A-21447). HRP-linked anti-rabbit IgG (NA934) and HRP-linked anti-mouse IgG (NA031) were from GE Healthcare Life Sciences.

Lungs were harvested and fixed as previously described (Frank et al., 2019 (link)). They were then washed, dehydrated, embedded in paraffin, and 6-µm sections were obtained. IHC was performed on E15.5, E17.5 and E18.5 sections. In brief, the sections were deparaffinized and rehydrated followed by antigen retrieval (Reveal Decloaker, MSPP-RV1000M, Biocare Medical). The sections were incubated with 3% H₂O₂ for 15 min to quench endogenous peroxidases, blocked with 5% donkey serum, and, finally, incubated with primary antibody (rabbit anti-VWF, 1:250, F3520, Sigma-Aldrich; goat anti-EMCN, 1:200, AF4666, R&D Systems; mouse anti-ACTA2, 1:500, A5228, Sigma-Aldrich; rabbit anti-RFP, 1:50, 600-401-379, Rockland Immunochemicals) in 0.1% PBST overnight at 4°C. The presence of relevant proteins was visualized using Alexa Fluor secondary antibodies (donkey anti-mouse Alexa Fluor 488, 1:250, A-21202, Sigma-Aldrich; donkey anti-rabbit Alexa Fluor 555, A-31572, Thermo Fisher Scientific; donkey anti-goat Alexa Fluor 647 1:250, A-21447, Thermo Fisher Scientific).

Eyes of 1 month old were fixed in 4% PFA for 3 h at 4°C. The bulbus was cut open along the ora serrata. The lens and remnants of the retina were removed. The cornea was cut open and the iris was carefully removed without harming the ciliary body, modified after Thomson and Quaggin (2018) (link). The tissue was additionally fixed in 100% methanol for 20 min at −20°C. Wholemounts were pretreated with 50 mM NH₄Cl for 1 h and 0.5% Triton X-100 for 30 min at RT. As blocking solution, we used 2% BSA, 0.2% CWFG, 0.1% Triton X-100 in 0.1 M phosphate buffer, which was applied for 1 h at RT. The first antibody goat anti-cluster of differentiation 31 (CD31) (1:100, R&D Systems; Cat# AF3628, RRID: AB_2161028) was incubated at 4°C over night. Afterwards, wholemounts were washed 3 times for 10 min with 0.1 M phosphate buffer and then incubated with donkey anti-goat Alexa 647 (1:1,000, Thermo Fisher Scientific Cat# A-21447, RRID: AB_2535864) for 2 h at RT. After washing 2 times with 0.1 M phosphate buffer and once with H2O a.d. for 10 min each, wholemounts were mounted on an object slide with mowiol (Carl Roth, Germany).