4g10 platinum

Primary fibroblasts were serum-starved for 18 h before treatment with 100 nM insulin for 5 min. Cells were lysed in 1× lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton; Cell Signaling) supplemented with 1× COMPLETE protease inhibitors (Roche) and 1× phosphatase inhibitors I and II (Sigma-Aldrich). Cell lysates containing 150 μg of total protein were incubated with 5 μg α-INSR antibody (Cell Signaling, 3025), or 5 μg α-IGF1R antibody (Cell Signaling, 3018) at 4°C overnight. Immunocomplex was pulled down using Protein A-Sepharose (GE Healthcare). Input and immunoprecipitation samples were analyzed by Western blot using α-phosphotyrosine antibody (1:1,000; Millipore, 4G10 Platinum), or the above α-INSR (1:1,000) and α-IGF1R (1:1,000) antibodies. Twenty percent of input lysates were loaded as a control.

Western blotting was performed essentially as described (8 (link)) using the primary antibodies PEAR1-EC antibody (R&D Systems), PEAR1-EC antibody (Santa Cruz Biotechnology), FcεR1α antibody (LifeSpan Biosciences), anti-PLCγ2 (Sigma), anti-PLCγ2-P (Cell Signaling Technology), anti-AKT-P (Cell Signaling Technology), and anti-phosphoprotein (Tyr(P)) 4G10 Platinum (Millipore). After adding horseradish peroxidase-conjugated secondary antibodies (Dako), immunoreactive bands were visualized by ECL (Amersham Biosciences). PEAR1 phosphorylation on Tyr residues, referred to as PEAR1-P, was evaluated after immunoprecipitation with PEAR1-EC antibody and detection of Tyr(P) by 4G10 Platinum as previously described (8 (link)).

For each immunoprecipitation, HeLa cells were lysed after reaching 80% confluency, using 500 μl ice-cold extraction buffer containing 20 mM HEPES, pH 7.5, 10 mM NaCl, 15 mM MgCl₂, 10 mM NaF, 1 mM EDTA, 1 mM Na₃VO₄, 0.5% (w/v) Nonidet-P-40, supplemented with protease and phosphatase inhibitors cocktail (Roche). After 30 min of cell lysis on ice in the extraction buffer, the samples were centrifuged at 6,000g for 30 min at 4 °C. The supernatants were then normalized to 500 μg and incubated under rotation at 4 °C with 1.3 μg anti-GFP antibody, as indicated for 2 h or O/N, before addition of protein A/G-Sepharose for a further 30 min. When using the anti-phospho-tyrosine antibody conjugated to the agarose matrix (4G10 Platinum, Millipore), the resin was first equilibrated in extraction buffer and then 20 μl of resuspended resin was used for each sample, incubating O/N on a rotating shaker. The beads were collected and washed twice with 500 μl ice-cold complete extraction buffer, and twice with 500 μl ice-cold extraction buffer without detergent. The western blots were visualized using the ECL reagents (GE Healthcare), according to the manufacturer's instructions for ECL-based detection. Where stated, band intensities were quantified using Quantity One software (Bio-Rad Laboratories). Full-scan images of all western blotting data are reported in Supplementary Fig. 6.