Whole cell lysates were prepared, and the assay was carried out according to standard procedures. Cells were washed twice with cold DPBS and lysed in lysis buffer containing 1 mM phenylmethylsulfonyl fluoride and a 1× protease inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged at 12000 rpm for 15 min at 4°C, and the supernatants obtained were used for analysis. The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal quantities of protein were loaded onto Bolt 4–12% Bis-Tris Plus Gels (Invitrogen) and separated at 200 V for 35 min. Proteins were transferred onto polyvinylidene fluoride membranes (Invitrogen) using a Power Blotter (Invitrogen). The membranes were blocked with EveryBlot blocking buffer (Bio-Rad, Hercules, CA, USA) for 15 min at 24°C. Membranes were incubated with the following primary antibodies for 1 h at 24°C: rabbit anti-CREB (1:1000), rabbit anti-phospho CREB (1:1000), mouse anti-ERK (1:2000), and rabbit anti-phospho ERK (1:2000). Primary antibodies were purchased from Cell Signaling Technology. Blots were washed six times for 5 min with Tris-buffered saline containing 0.05% Tween 20 and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies (1:2000, Thermo Fisher Scientific) for 1 h at 24°C. Immunoreactivity was detected with Amersham ECL (Cytiva, Marlborough, MA, USA) using an iBright 1500 (Invitrogen). The intensity of the protein bands was quantified using iBright analysis and normalized to β-actin in each sample.
Free full text: Click here
