Anti hnrnpu antibody

Biotinylation of miRs was performed by use of the Pierce™ RNA 3ʹ-End Desthiobiotinylation Kit (Thermo Fisher Scientific, Cat. # 20163) according to the manufacturer’s protocol. Biotinylated miRs were purified with an miRNeasy Mini Kit (Qiagen, Cat. # 217004). 50 pmol of biotinylated miRs were conjugated with 50 μL of Dynabeads M-280 Streptavidin by using 1× RNA Capture Buffer (provided with Pierce™ Magnetic RNA-Protein Pull-Down Kit, Cat. # 20164) according to the manufacturer’s protocol. Total cellular extracts were collected with Pierce IP Lysis Buffer (Life Technologies Cat. # 87787). Then, 1 mg of cellular extract was pre-cleared with 20 μL of Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, Cat. # 11205D). The biotin-RNA-streptavidin complex was incubated with 1 mg of pre-cleared cellular extract at 4°C for 1 h. After washing five times with washing buffer (150 mM KCl; 25 mM TRIS-HCl at pH = 7.4; 5 mM EDTA; 0.5 mM DTT; 0.5% NP40; and cOmplete Protease Inhibitor Cocktail (Roche, Cat. # 4693132001), the bound proteins were eluted with 50 μL of Biotin Elution Buffer after incubation for 30 min at 37°C with agitation. The entire elution volume was loaded onto a 4–15% polyacrylamide precast protein gel (Bio-Rad, Cat. # 456-1084). Subsequently, immunoblotting for hnRNPU using an anti-hnRNPU antibody (Abcam, Cat. # ab10297) was performed as described above.

Immunocytochemistry of endothelial cells was performed by using anti-hnRNPU antibody (Abcam) and Atto 565 phalloidin (Sigma-Aldrich Cat. # 94072). 3 × 10⁴ HCAECs per well were grown in a 4-well chamber slide. 24 h after seeding, cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at RT. Fixed cells were washed with PBS and incubated with 0.25% Triton X-100 in PBS for 10 min at RT for permeabilization of the cell membranes. After three washing steps with PBS, cells were incubated with the blocking solution (0.25% Triton X-100; 1% bovine serum albumin [BSA] in PBS) for 1 h at RT. Subsequently, cells were incubated with anti-hnRNPU antibody 1:500 in the blocking solution overnight at 4°C. After extensive washing with PBS, the cells were incubated with Alexa Fluor-488 conjugated secondary antibody (Thermo Fisher Scientific, Cat. # A28175, 1:1000) and Atto 565 phalloidin (Sigma-Aldrich) for 60 min at RT. After washing with PBS, DAPI staining was applied and the chamber slide was mounted by using ProLong Gold Antifade (Invitrogen, Cat. # 502081). Images were taken with a Zeiss Axio Observer inverted microscope and analysed with the ZEN 2.3 pro software.