Mouse grb2

Fifteen to twenty μg of total protein of each sample was subjected to SDS-PAGE/Western blotting. Specific proteins were detected with antibodies against mouse GRB2 (BD Pharmingen, 610111), mouse RSK2 (Santa Cruz, Heerhugowaard, The Netherlands, SC-9986), rabbit ERK1/2 (Cell signaling, Leiden, The Netherlands, #9102), rabbit RSK1 (Santa Cruz, SC-231), rabbit CCND1 (Thermoscientific, Etten-Leur, The Netherlands, RM-9104-S) and mouse anti-β-actin (Sigma, A5441). Secondary HRP-conjugated antibodies used were Goat-anti-mouse (Santa Cruz, SC-2005) and Goat-anti-rabbit (Jackson ImmunoResearch, Westgrove, PA, USA, 111-035-144).

Following cell adhesion, coverslips were washed twice with PBS and fixed with 4% paraformaldehyde pH 7.4 (BioShop) for 15 minutes at room temperature. After three washes with PBS, fixed cells were permeabilized using 0.2% Triton X-100 (Sigma-Aldrich) for 15 minutes at room temperature. Coverslips were blocked in PBS supplemented with 10% normal goat serum (Wisent) and 0.1% NP40 (Sigma-Aldrich) for 1 hour at room temperature and incubated with the following antibodies diluted in blocking solution: mouse FLAG (Sigma-Aldrich), mouse GRB2 (BD Biosciences), rabbit GRB2 (Santa Cruz) or rabbit MPZL1 (Cell Signalling Technology) for 1 hour at room temperature and then overnight at 4 °C. After washes in 0.1% NP40 in PBS, coverslips were incubated with Alexa 568-conjugated goat anti-rabbit (Invitrogen) or Alexa 488-conjugated goat anti-mouse (Cell Signalling Technology) antibodies for 1 hour at room temperature. They were washed three times with 0.1% NP40 in PBS and twice with PBS before being mounted on slides using ProLong Gold antifade with DAPI (Thermo Fisher). Pictures were acquired with an Olympus FV1000 using the FluoView software or with a Nikon Eclipse E600 imaging system using MetaView.

Proteins were resolved on 8 to 15% polyacrylamide gels and transferred onto nitrocellulose membranes (GE Healthcare). Loading was verified with Ponceau S (Sigma-Aldrich) staining. Membranes were blocked in 5% non fat milk in TBS for 1 hour at room temperature before being incubated with antibodies overnight at 4 °C in TBS-T supplemented with 5% BSA or 1% non fat milk according to manufacturer recommendations. Antibodies used were as follows: mouse GRB2 (BD Bioscience), rabbit PTPN11 (Santa Cruz), rabbit MPZL1 (Cell Signalling Technology), mouse tubuline (Cell Signalling Technology) or goat dynamin 2 (Santa Cruz). Membranes were washed with TBS-T before being incubated for 1 hour at room temperature with the following HRP-linked antibodies diluted in 1% milk in TBS-T: anti-FLAG M2 (Sigma-Aldrich), horse anti-mouse IgG (Cell Signalling Technology), goat anti-rabbit (Cell Signalling Technology) or rabbit anti-goat IgG (Thermo). Signal was revealed using BioRad Clarity Western ECL substrate and detected either on Hyblot CL autoradiography films (Denville) or with an Amersham Imager 600RGB (GE Healthcare). Signal quantification was performed using Image J software gel analysis tools (NIH).