Antibodies for erk

Briefly, 10 × 10⁶ cells were collected and lysed in 1 ml of RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for immunoprecipitation. Cell extracts were pre-cleared by mixing lysates with 2 μg of normal control IgG and 50 μl of protein G agarose and incubating for 4 h at 4 °C. The pre-cleared lysates were mixed with 2 μg of the appropriate antibodies plus 20 μl of protein G agarose beads and rotated overnight at 4 °C. The immunoprecipitated complexes were obtained by centrifugation, washed three times with RIPA lysis buffer and boiled at 75 °C in loading buffer for 10 min for western blot. Cell lysates were also directly used for western blot analysis. All western blot photos were representative photos from at least two experiments with similar results. Antibodies for HSC 70 (cat no. SC-7298), Lyn (cat no. SC-7274), Fyn (cat no. SC-16), PSTPIP2 (cat no. SC-83015), GATA1 (cat no. SC-1234), SHC (cat no. SC-1695) were purchased from Santa Cruz Biotechnology. Antibody for FLAG (cat no. F3165) was obtained from Sigma. Antibodies for ERK (cat no. 4695), p-ERK (cat no. 4370), p-SRC (cat no. 2101) were purchased from Cell Signaling Technology.