Skip to main content
. 2012 Nov 29;288(4):2210–2222. doi: 10.1074/jbc.M112.397398

FIGURE 6.

FIGURE 6.

Netrin-1 induces CREB phosphorylation. A, U87MG cells were treated with netrin-1 (200 ng/ml). After 30 min, cell lysates were collected and applied to a phosphoantibody array as described under “Experimental Procedures.” B, U87MG cells were treated with netrin-1 for 30 min at the indicated dose (upper panels). Cells were treated with netrin-1 (400 ng/ml) and lysates were collected at the indicated time points and Western blotted with anti-CREB or anti-ERK antibody (lower panels). The intensity of phosphorylated CREB and ERK bands was normalized to their respective total CREB and ERK, and the numbers below gel lanes represent the fold-change in intensity relative to control. C, U87MG cells were treated with U0126 (10 μm) prior to 30 min of netrin-1 treatment. Cells were collected and analyzed by Western blotting. The intensity of phosphorylated CREB bands was normalized to their respective total CREB and the numbers below gel lanes represent the fold-change in intensity relative to control. D, U87MG cells were transfected with control or CREB-specific siRNA (#1 and #2) (20 nm). After 24 h, the silencing effect of CREB siRNA on CREB protein was analyzed by Western blotting. The intensity of CREB bands was normalized to their respective β-actin controls and the numbers below gel lanes represent the fold-change in intensity relative to controls. E, U87MG cells were transfected with control or CREB-specific siRNA (#1 and #2) (20 nm). After 24 h, cells were treated with netrin-1 (400 ng/ml) and assessed for their ability to invade through Matrigel-coated transwells. Data represent the mean ± S.D. (n = 3), *, p < 0.05. F, U87MG cells were transfected with control or CREB-specific siRNA (20 nm). Spheroids were stimulated with netrin-1 (400 ng/ml) and stained with Alexa Fluor 488-phalloidin and DAPI and assessed for total sprout length and sprout number. Data represent the mean ± S.D. (n = 3), *, p < 0.05. The scale bar indicates 100 μm. G, U87MG cells were transfected with control or CREB-specific siRNA (#1 and #2) (20 nm). Cells were treated with netrin-1 (400 ng/ml) for 2 h and CatB mRNA levels were analyzed by qRT-PCR. Data represent the mean ± S.D. (n = 3), *, p < 0.05.