Fig 2: Localization of endogenous Gal-3 in ADSCs and HUVECs. (A) Immunofluorescence of Gal-3 in ADSCs and HUVECs in 1-day-old cultures. Cells were stained for Gal-3 present in the cell plasma membrane (green fluorescence) and for intracellular Gal-3 (red fluorescence). For Gal-3 staining, an anti-Gal-3 antibody produced in rabbits (Sigma-Aldrich, Cat. No. SAB4501746) was used. Orthogonal projections depict the localization of Gal-3 in the cell plasma membrane or the intracellular space. Andor Dragonfly 503 scanning disc confocal microscope; Zyla 4.2 PLUS sCMOS camera; objective HC PL APO 40×/1.10 W CORR CS2; scale bar 50 µm. (B) Western blot of Gal-3 expression in the cytosol of ADSCs and HUVECs. In both tested cell types, a Gal-3 band was detected at a size of 28 kDa.

Fig 3: Super-resolved image of HA molecules 24 h post transient D2HA transfected NIH3T3 cells for 5 ms, 30 ms and 50 ms exposed acquired data using Zyla 4.2 plus (Andor). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2\times 2$$\end{document}2×2 binning is used while acquiring data. Each reconstructed image contains approximately, 20, 000 HA molecules. Corresponding transmission and fluorescence (at, 510 nm) images are also shown.

Fig 4: Detection of NCK1/2 transgenes in ARPE-19 cells stably engineered with pSBtet-BP-transgene plasmids.ARPE-19 cells were subjected to transfection to generate cells that stably carry the transgene for inducible expression of eGFP-tagged NCK1 or 2, as in Procedure B. Then, induction of NCK1-eGFP (A) or NCK2 -eGFP (B) was performed as in Procedure C, using doxycycline concentrations (ng/mL) as indicated for 24 h. (A) Whole-cell lysates of NCK1-eGFP stable cells were resolved by SDS-PAGE followed by transfer to PVDF membranes and then immunoblotted using antibodies as indicated. Also shown are the approximate molecular weights (kDa) for each blot panel. (B) Cells were subjected to PFA fixation, and DAPI labeling using standard fluorescence microscopy sample preparation, and then imaged using a Quorum Diskovery instrument comprised of a Leica DMi8 microscope. Imaging was done using a 40× objective with 405- and 488-nm laser illumination, 450/55 and 525/50 emission filters, and acquired using a Zyla 4.2Plus sCMOS camera (Andor). Scale bars: 40 μm.