Ibright cl1000 machine

Whole cell lysates were prepared using radio immunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 50 mM Tris HCl, 1 mM EGTA, 0.24% (w/v) sodium deoxycholate,1% (w/v) Igepal, pH 7.5) with protease and phosphatase inhibitors. Inhibitors including NaF (1 mM) and Na₃VO₄ (1 mM), aprotinin (1 μg/ml), leupeptin (1 μg/ml), pepstatin (1 μg/ml), and phenylmethylsulfonylfluoride (PMSF, 1 mM) were added fresh, just prior to lysis. Total protein lysates (20 μg) were separated on SDS polyacrylamide gradient gels (4–12%) and transferred to polyvinylidene difluoride (PVDF) membranes, as described previously^{15 (link)}. Membranes were blocked with milk (5%, w/v) diluted in tris-buffered saline containing tween-20 (TBS-T, 0.01%, v/v). Blots then were incubated ON at 4 °C with the appropriate primary antibody. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) (Cell Signaling) at RT for 1 h with chemiluminescent detection using ECL Plus substrate (Amersham Biosciences, Piscataway, NJ). Images were developed and acquired with an iBright CL1000 machine (Applied Biosystems).

Whole cell lysates were prepared using radio immunoprecipitation assay (RIPA) lysis buffer (150 mmol·L⁻¹ NaCl, 50 mmol·L⁻¹ Tris HCl, 1 mmol·L⁻¹ EGTA, 0.24% sodium deoxycholate,1% Igepal, pH 7.5) containing NaF (25 mmol·L⁻¹) and Na₃VO₄ (2 mmol·L⁻¹). Aprotinin, leupeptin, pepstatin, and phenylmethylsulfonylfluoride (PMSF) were added fresh, just prior to lysis. Whole cell lysates (20 μg) were separated on polyacrylamide gels (4%–12%) and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with milk (5%, w/v) diluted in TBS-T. Blots then were incubated overnight at 4 ˚C with the appropriate primary antibodies. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:5 000 dilution) (Cell Signaling) at RT for 1 h. ECL plus was used to detect chemiluminescence (Amersham Biosciences, Piscataway, NJ). Images were developed and acquired with an iBright CL1000 machine (Applied Biosystems), and densitometry was determined using ImageJ software version 1.45s (NIH).