Fuji las 4000 mini luminescent image analyzer

The anchorage-independent conditions were used to perform all the signaling assays. Wells of microtiter plates were coated with 1.2 mg/cm² Poly(2-hydroxyethyl methacrylate) (poly-HEMA) following the protocol as described [20 (link)]. Cells were cultured in normal conditions using regular tissue culture plate until 80–90% confluence in DMEM 10% FBS, 1% antibiotics and 1% non-essential amino acids at 37°C in 5% CO₂ atmosphere. The cells were collected and plated in poly-HEMA plate for starvation for 4 hrs in DMEM without FBS. Serum-starved cells were treated with WT IGF2 or IGF2 mutants (R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E). After treatment, cells were collected by spinning the samples at 3.5 rpm and then solubilized using lysis buffer (20 mM HEPES pH 7.4, 10% glycerol, 100 mM NaCl, 1 mM MgCl₂, 1% Nonidet P-40, 20 mM NaF, 1 mM PMSF, protease inhibitor mixture (Sigma-Aldrich) and 1 mM Na₃VO₄). BSA assay (Thermo Scientific) was performed to determine the protein concentration to each sample. The cell lysates were analyzed by western blotting using different antibodies. The HRP-conjugated anti-IgG antibody, Supersignal West Pico and Femto (Thermo Scientific) were used to detect IgG. Fuji LAS 4000 mini luminescent image analyzer and Multi Gauge V3.0 software (Fujifilm, Tokyo, Japan) were used for analysis of the images.

Human umbilical vein endothelial cells (HUVECs) were starved for 4 h in M200 basal medium without low serum growth supplement and then treated with VEGF165 wild-type or mutant protein (10 ng/mL) for 10 min before lysis and Western blot analysis. Cells were lysed in lysis buffer 20 mM HEPES (pH 7.4), 100 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM MgCl₂, 1 mM PMSF, 20 mM NaF, 1 mM Na₃VO₄, and protease inhibitor mixture (Sigma-Aldrich). Cell lysates were analyzed by Western blotting using specific antibodies directed to ERK1/2 (p44/42) or phospho ERK1/2 p44/42 (Thr202/Tyr204). Bound IgG was detected using the HRP-conjugated second antibody and SuperSignal West Pico (Thermo Fisher Scientific). Images were evaluated using a Fuji LAS 4000 mini luminescent image analyzer and Multi Gauge v.3.0 software (Fujifilm, Tokyo, Japan).