After the incubation period, cells were washed twice with warmed medium, followed by 5 min incubation with medium.
Following 30 min incubation, the medium was replaced with new complete RPMI containing 10 µg/ml gentamicin for the rest of the experiment.
The cells were starved in serum free medium for 3 h and treated with different concentrations of cFΦR4-PTP1B or untagged PTP1B for 2 h, followed by a 30 min incubation in medium supplemented with 1 mM sodium pervanadate.
The dissociated heart tissue was then incubated in medium containing 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 12.5 μM CaCl2 for 10 min at RT, followed by an additional 10 min incubation in medium containing 5% fetal bovine serum and 12.5 μM CaCl2.
Following 30 min incubation, the medium was removed and 150μl of HBSS, 100μl of 5-amino-2.3-dihydro-1.4-phthalazinedione (Luminol, Sigma-Aldrich, 1mM) and a phorbol ester, 12- O-tetradecanoyl-phorbol-13-acetate (TPA) 1mM (Sigma-Aldrich) was then added for 30 minutes.
After incubation at 37°C for different time points up to 60 min, the incubation medium was removed, and cells were rinsed with 250 μL of unsupplemented medium.
After 15 min, the incubation medium was substituted for the same solution containing collagenase only.
After 90 min incubation, the transfection medium was removed and replaced with fresh medium.
After 24 hours of incubation with the desired treatments, cells were washed and incubated with HEPES buffer (5 mM HEPES containing (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2 and 10 glucose, pH adjusted to 7.4), for 120 min. Then, incubation medium was collected, culture wells were washed with PBS, and adherent cells were collected in 0.5 N NaOH solution for protein determination [18].
After an additional 45 min incubation period, the medium was removed.
