Swine anti rabbit horseradish peroxidase conjugated antibodies

SDS lysed tissue extracts were subjected to Western blotting as described previously^{18 (link)}. Blots were incubated overnight at 4 °C with primary antibodies diluted in Starting block (Pierce, Chester, UK). Antibodies were used at the following concentrations: N-cadherin (BD Biosciences: 610920, 1:2,500), β-actin (Sigma, A5316, 1:10,000). Bound antibodies were detected by rabbit anti-mouse or swine anti-rabbit horseradish peroxidase conjugated antibodies (Dako) and enhanced chemiluminescence (Amersham International).

SDS lysed cell extracts were subjected to Western blotting as described previously [5] (link). Blots were incubated overnight at 4 °C with primary antibodies diluted in 5% BSA/TBS. Antibodies were used at the following concentrations: N-cadherin (BD Biosciences: 610920, 1:2500), GAPDH (Chemicon: MAB374, 1:5000), and FGFR1 (R&D Systems: MAB658, 1:50). Bound antibodies were detected by rabbit anti-mouse or swine anti-rabbit horseradish peroxidase conjugated antibodies (Dako) and enhanced chemiluminescence (Amersham International). Stain free gel (BioRad) loading controls were used in some experiments. Fc tag was detected using the goat-anti-mouse secondary antibody (diluted 1:1000 in 5% Marvel in TBS-T).
Immunoprecipitation of GFP tag was performed as follows. FGFR1-GFP was overexpressed in full media using AMAXA (following the manufacturer's instructions) in 4 T25 flasks. After 24 h cells were treated with nothing (control), 200 pM Fc, SNC-Fc or SNC-Fc FGFR1mut in serum free media for 1 h. Cells were lysed in 400 μl RIPA buffer with complete protease inhibitor (Roche). Lysates were incubated on a rotator at 4 °C with GFP-agarose beads (abcam: ab69314) overnight, centrifuged and washed twice with RIPA buffer. The beads were resuspended in 30 μl Laemmli buffer (BioRad), and boiled at 95 °C for 5 min before loading onto a Western blot as described above. Fc tag detection was used.