Anti human β actin

Whole cell lysates were subjected to Western blotting as previously described (28 (link)). p-Ser473-Akt, GSK3β, phospho-GSK3β-Ser9, ERK1/2, and p-ERK1/2 antibodies were purchased from Cell Signaling Technology (Herts, UK). Anti-human β-actin was obtained from Santa Cruz Biotechnology and anti-human GAPDH antibody from Abcam.

HDBECs were lysed in NuPAGE LDS Sample buffer under reducing or nonreducing conditions, according to the primary antibody manufacturer’s recommendations. The samples were separated onto a 4%–12% Bis-Tris gel (Life Technologies) and blotted to a nitrocellulose membrane (Immobilon). To analyze proteins secreted or cleaved from endothelial cells, conditioned medium derived from HDBECs maintained for 24 hours in EBM media supplemented with 1% FBS was collected, cell debris were removed by centrifugation, and 25 μg of total protein was separated onto a gel. The membranes were blocked and probed with anti–human CD93 (HPA, HPA009300), anti–human β₁ integrin (K20, Santa Cruz Biotechnology, sc18887), anti–human CD93 (MBL Life Science, D198-3), anti–human MMRN2 (Santa Cruz Biotechnology, sc54021), or anti–human β₁ integrin (12G10, Abcam, ab30394) or with anti–human β-actin (Santa Cruz Biotechnology, sc-1615) followed by appropriate horseradish peroxidase–labeled secondary antibodies. A representative blot image of at least 3 independent experiments is shown.

RIPA buffer was used for lysis of cultured cells. The protein concentration in samples/lysates was measured using The Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Total protein lysates (25 µg for TPC1 cell line; or 30 µg in the case of FTC133 and BCPAP cell lines) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, and immunostained overnight at 4 °C, using the antibody of interest. Anti-NOP53 (1:1.000, #73225) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-human β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. Membranes were incubated with appropriate secondary horseradish peroxidase conjugated IgG (anti-rabbit 1:3.000, Cell Signaling Technology; or anti-mouse 1:3.000, Santa Cruz Biotechnology). To prevent non-specific background binding of the primary and/or secondary antibodies to the membrane, we used 5% Bovine serum albumin (BSA) in 10× Tris Buffered Saline (TBS) with 0.1% Tween-20 for 60 min, as a blocking solution. Proteins were detected using enhanced chemiluminescence (ECL, Pierce Biotechnology, Waltham, MA, USA).