Phosphatase inhibitor cocktail 3

293T cells were cultured to approximately 50% confluence in 10 cm plates and then cotransfected with 2 μg pcDNA6-N-3XFLAG-Lrp6 (123595, Addgene) and 2 μg mSELENOP plasmids (ref. 62 (link) and the present study) with polyethylenimine. Forty-eight hours later, cells were incubated on ice for 10 minutes in FLAG IP Lysis Buffer (L3412, MilliporeSigma) with phosphatase inhibitor cocktail 2 (P5726, MilliporeSigma), phosphatase inhibitor cocktail 3 (P0044, MilliporeSigma), and protease inhibitor cocktail (P8340, MilliporeSigma), and then transferred into microcentrifuge tubes and centrifuged at 16,000g for 10 minutes at 4°C. Supernatant protein concentrations were quantified with a BCA Protein Assay Kit (23225, Pierce, Thermo Fisher Scientific). Total protein (2 mg) was used for IP with ANTI–FLAG M2 Affinity Gel (A2220, MilliporeSigma) according to the manufacturer’s protocol. Bound proteins were eluted with 150 ng/μL 1x FLAG Peptide (F3290, MilliporeSigma) at 4°C for 30 minutes.

Total protein from 100 mg of 4-day-old seedlings was extracted in 300 μl of extraction buffer containing 100 mM Tris-Cl pH 7.5, 100 mM NaCl, 5 mM EDTA, 5% SDS, 20% glycerol, 20 mM DTT, 40 mM β-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 40 μM MG115, 40 μM MG132, 10 mM N-ethylmaleimide, 1× phosphatase inhibitor cocktail 3 (MilliporeSigma, Burlington, MA), 1× EDTA-free protease inhibitor cocktail (MilliporeSigma, Burlington, MA), and 0.01% bromophenol blue. Samples were immediately boiled for 10 min and centrifuged at 16,000 × g for 10 min. Cleared protein samples were separated via SDS-PAGE, transferred to nitrocellulose membranes, probed with the indicated primary antibodies, and then incubated with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Bio-Rad Laboratories, 1706515 for anti-rabbit and 1706516 for anti-mouse). Primary antibodies, including polyclonal rabbit anti-PIF4 antibodies (Agrisera, AS12 1860) and monoclonal mouse anti-actin antibodies (Sigma-Aldrich, A0480), were used at a 1:2000 dilution. The signals were detected via a chemiluminescence reaction using SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific, Waltham, MA).

Whole cell extracts were prepared by lysing the cells or emboli with RIPA buffer (25 mM Tris [pH 8.0]; 50 mM NaCl; 1 mM EDTA; 0.5% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 10 μL/mL protease inhibitor cocktail, MilliporeSigma, P8340; 10 μL/mL phosphatase inhibitor cocktail #2, MilliporeSigma, P5726; 10 μL/mL phosphatase inhibitor cocktail #3, MilliporeSigma, P0044). Tumor extracts were prepared as described (27 (link)). Protein concentrations were measured by BCA assay (Thermo Fisher Scientific, 23225). About 10–20 μg protein was loaded onto NOVEX WedgeWell 4%–20% Tris-glycine gels, and Western blot analyses were carried out as described (79 (link)). Representative data are shown and were repeated at least 3 times each.

Cells were lysed on ice in 50 mM HEPES (pH 7.5), 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, 2.5 mM sodium orthovanadate, 1 mM PMSF and 1% of phosphatase inhibitor cocktail 3 (MilliporeSigma, St. Louis, MO, USA). Lysates were sonicated 1 min on ice and centrifuged at 22,000 G for 15 minutes at 4 °C. The supernatant was collected and syringe-filtered through a 0.2 mm SFCA membrane. The filtered lysate (approximately 1.5 mg of protein per sample) was brought to 1 M NaCl and precleared by incubating lysate with 500 µl Sepharose CL-4B for 15 min and then incubated with 100 µL multiplexed inhibitor-conjugated beads (MIBs) consisting of Sepharose-conjugated Bisindoylmaleimide-X, dasatinib, Purvalanol B, PP58 and VI16832. The MIBs were washed three times with high-salt and low-salt buffers (50 mM HEPES (pH 7.5), 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, and 10 mM sodium fluoride, and 1 M NaCl or 150 mM NaCl, respectively). Proteins were eluted from MIBs with elution buffer containing 7 M urea, 2 M thiourea, 4% CHAPS and 40 mM DTT.

Cells were lysed in 1x RIPA buffer (9806 Cell Signaling) containing 10 mM PMSF, Protease Inhibitor Cocktail (M250 Amresco), Phosphatase Inhibitor Cocktail 2 (P5726 Milipore), and Phosphatase Inhibitor Cocktail 3 (P0044 Milipore) followed by immunoblotting using LI-COR Odyssey® CLx Imaging System. Antibodies were typically duplexed using rabbit antibodies for phosphorylated antibodies and mouse antibodies for total protein. Li-Cor secondary antibodies, Goat anti-Rabbit IRDye 680RD and Goat anti-Mouse IRDye 800CW, were used with the duplexed primary antibodies.
All primary antibodies were rabbit unless specified and were sourced as follows: PTPRS (mouse Cat.No.ab55640 Abcam); PTPRS (goat AF3430 R&D Systems); alpha-Tubulin (mouse sc-8035 Santa Cruz). All other antibodies were obtained from Cell Signaling: phospho-Erk1/2 T202/Y204 (Cat.No.4370); phospho-Erk1Y204/Erk2 Y187 (mouse D1H6G); Erk1/2 (mouse 4696); phospho-MEK1/2 S217/221 (9154); MEK (mouse 4694); phospho-EGFR Y1173 (4407); EGFR (mouse 2239); Elk-1(rabbit Ab 9182), p-Elk1 (Ser383 mouse Ab 9186), MSK1 (rabbit Ab 3489), p-MSK1 (Thr 581 rabbit Ab 9595) and Erk Rabbit Ab 4695).
Active Ras assay was performed using the Active Ras Pull Down and Detection kit from Thermo Fisher (Cat.No.16117).

Cell lysates were obtained using 1x RIPA buffer (9806 Cell Signaling) containing 10 mM PMSF, Protease Inhibitor Cocktail (M250 Amresco), Phosphatase Inhibitor Cocktail 2 (P5726 Millipore), and Phosphatase Inhibitor Cocktail 3 (P0044 Millipore). The LI-COR Odyssey^® CLx Imaging System was used to image all immunoblots. Antibodies were typically duplexed using rabbit antibodies for phosphorylated antibodies and mouse antibodies for total protein. Li-Cor secondary antibodies, Goat anti-Rabbit IRDye 680RD and Goat anti-Mouse IRDye 800CW, were used with the duplexed primary antibodies.
Rabbit primary antibodies were used unless specified and were sourced as follows: PTPRS (goat AF3430 R&D Systems); alpha-Tubulin (mouse sc-8035 Santa Cruz). All other antibodies were obtained from Cell Signaling: phospho-Erk1/2 T202/Y204 (Cat.No.4370); phosphor-Akt S473 (mouse 4051); Akt (4691); phosphor-src Y416 (2101) and Src (mouse 2105).

Cells were lysed following the respective treatments in cell lysis buffer containing 50 mM Tris pH 7.4, 120 mM NaCl, 5 mM EDTA, 0.5% NP40 and 1 mM DTT, supplemented with the phosphatase inhibitor cocktail I, II (Sigma) and complete protease inhibitor tablet (Roche Diagnostics, Indianapolis, IN, USA). For immunoprecipitation, cell lysis was prepared in the above cell lysis buffer except that NP40 was reduced to 0.1%.