β actin 8h10d10

Cell lysates were prepared using 1× radioimmunoprecipitation assay (RIPA) lysis buffer (Dynebio, Korea), supplemented with a protease inhibitor (Roche, Switzerland), cleared by centrifugation at 12,000 × g, separated by SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Primary antibodies were incubated overnight at 4°C in 1× Tris-buffered saline with Tween 20 (TBST) with 5% skim milk or 5% BSA, E7 (8E2, 1:1,000; Abcam, UK), and β-actin (8H10D10, 1:1,000; Cell Signaling Technology, USA).

Serum starved HUVEC (∼70–80% confluent) were treated with HKE₂ (50–500 nM), VEGF₁₆₅ (50 ng/ml), or vehicle (ethanol, 0.1% v/v). After 0.5 to 2 h, cell lysates were analyzed by SDS-PAGE followed by Western blot for levels of phosphorylated tyrosine, phosphorylated and total VEGFR2, phosphorylated and total ERK 1/2, Akt, and p38 MAPK, and β-actin. Primary antibodies used were as follows: phospho-tyrosine (P-Tyr-100), phospho-VEGFR2 (Tyr1175) (19A10), VEGFR2 (55B11), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11), p44/42 MAPK (Erk1/2) (137F5), phospho-Akt (Ser473) (D9E) XP, Akt, phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP p38 MAPK, β-actin (8H10D10) (all from Cell Signaling Technology), and PTP1B (FG6-1G) (Calbiochem). Membranes were incubated with primary antibody at 4 °C overnight and subsequently incubated with secondary antibody, IRDye 680LT goat anti-rabbit (926-68024) or IRDye 800CW donkey anti-goat (926-32214) (LI-COR Biosciences) (1:10,000 dilution), at room temperature for 1 h. Membranes were scanned by Li-Cor Odyssey Infrared Imaging System. Bands were quantified using ImageJ software. The level of phosphorylated and total protein in each group was expressed as the ratio between phospho-/total-protein. Three or four independent experiments were performed.

For protein extraction, cells were lysed on ice by RIPA lysis buffer (KeyGEN), and the BCA Kit (KeyGEN) was used to determine the protein concentration. Similar amounts of protein were loaded on SDS–PAGE gels and subjected to electrophoresis, then transferred onto a PVDF membrane and blocked with Tris-buffered saline with 0.1% (vol/vol) Tween 20 (TBST) containing 5% (wt/vol) BSA for 2 hrs. Then, membranes were washed by TBST 3 times and incubated overnight at 4°C with primary antibodies against FAM111B (PA5-58474; Invitrogen), BAG3 (AB47124; Abcam), BCL2 (TA806639S; Origene), β-actin (8H10D10; Cell Signaling Technology) at a 1:1,000 dilution in 5% BSA. After washing by TBST 3 times, PVDF membranes were incubated with the appropriate fluorescent secondary antibody (1:10,000; Odyssey) for 2 hrs at room temperature. Proteins were visualized by Odyssey two-color infrared fluorescence imaging system (LI-COR Company, USA), cropped with Photoshop CS3 (Adobe Systems Incorporated,
San Jose, CA, USA). All experiments were independently repeated at least three times.