Anti phospho s6 kinase

Primary antibodies used in this study were anti-beta actin (Abcam, ab8224), anti-ERK1/2 (Cell Signaling, 9102), anti-phospho ERK1/2 (Cell Signaling, 9101), anti-lamin A/C (Santa Cruz, sc-7292), anti-lamin B1 (Santa Cruz, sc-30264), anti-S6 kinase (Cell Signaling, 2317), anti-phospho S6 kinase (Cell Signaling, 2211) and anti-LAP1 (Atlas antibodies, HPA050546).

The following antibodies were used for Western blotting: anti-phospho ERK 1,2 (Sigma St Louis, MO, Reference M8159)), anti-phospho Akt, anti-Akt and anti-phospho S6 Kinase (Cell Signaling, Cambridge, UK, respectively References 4051, 9272, 9234) and anti-TFE3 anti ERKs (Santa Cruz Biotechnology, Santa Cruz, CA references sc 5958 (TFE3) and sc 93 (ERK)).

To detect phosphorylation of c-RAF, and p70S6 kinase 3.10⁵ cells per well 6 wells plate are seeded in 2 ml of complete medium 24 h then cultured overnight in starvation medium. Medium is removed and cells are activated with cytokine at 20 ng/ml (1 ml) in free FCS RPMI. After an incubation of 20 min at 37 °C, the cells were lysed in 1% Triton × 100 lysis buffer, incubated for 1h on ice and centrifuged at 4 °C for 10 min at 10,000 g. The supernatants were collected and protein concentration was determined using the Bradford Assay (Bio-Rad, Marnes la Coquette, France). Proteins (70 μg) were resolved in 8% SDS–PAGE and transferred on nitrocellulose membrane. The membrane was blocked for 1 h at room temperature, by using 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween20 (Sigma–Aldrich) and incubated overnight at 4 °C with a anti-phospho-c-RAF antibody (1:1,000), anti-phospho S6 kinase (1:1,000) or with an anti-Actin antibody (1:1,000) (Cell Signaling, Leiden, Netherlands) used as a loading control, in blocking solution. After 3 washes, the membrane was incubated 1h at room temperature with goat anti-rabbit or goat anti-mouse secondary antibodies (1:10,000) (Jackson Immunoresearch, West Grove, USA) conjugated to horseradish peroxydase.