P fak y397

Cells were lysed in RIPA buffer containing protease inhibitors and protein lysates were subjected to SDS–PAGE and immunoblot analysis as previously described [34 (link)]. Antibodies used include pSTAT5 (Cell Signaling #9314, 1:1000), total STAT5 (Cell Signaling #9363, 1:1000), β-tubulin (Cell Signaling # 2146S, 1:1000), pFAK Y397 (Cell Signaling, # 3283S, 1:1000), and total FAK (Cell Signaling #3285S, 1:1000).

Cultured cells were homogenized in RIPA buffer (Cell Signaling) containing protease inhibitors (Complete; Roche, IN, USA). Protein concentrations were measured using the Bradford protein assay (Bio-Rad), according to the manufacturer’s instructions. Protein samples (5 µg) was boiling for 5 min, then subjected to 13- or 17-wells of 5%–20% SDS-polyacrylamide Supersep Ace (Wako) gel electrophoresis. Gels that contains separated products were transferred to nitrocellulose membranes, followed by blocking with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. Nitrocellulose membranes then incubate overnight with primary antibodies for p-FAK (Y397) (1:1000, Cell Signaling), FAK (1:1000, Cell Signaling), p-ERK1/2 (T202/Y204) (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), p-Akt (S473) (1:1000, Cell Signaling), Akt (1:1000, Cell Signaling), and GAPDH (1:1000, Proteintech, IL, USA). After washing with Tris-buffered saline containing 0.1% Tween 20, membranes were incubated with secondary antibody for anti-rabbit or anti-mouse IgG horseradish peroxidase-conjugated (1:3000, Cell Signaling) for one hour at room temperature. Bands of target proteins were detected using an ECL detection system (Wako). ImageJ Fiji (NIH) was used to analyses the bands. Data are showed as the mean ± standard error of the mean (SEM). GAPDH was used as the loading control.