Phosphatase inhibitor 2

Cells were lysed using lysis buffer (iNtRON Biotechnology, Seoul, Korea) with protease inhibitor cocktail (Biomake B14001, Houston, TX, USA), phosphatase inhibitor 2 (Sigma P5726) and phosphatase inhibitor 3 (Sigma P0044). Lysate proteins were quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (20 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes.
Membranes were blocked in 5% skimmed milk for 1 h and washed with Tris-buffered saline with Tween 20 (TBS-T). Membranes were immunoblotted with primary antibodies (1:1000) overnight at 4 °C and washed with TBS-T buffer. Subsequently, membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (1:2000) at room temperature for 6 h and washed with TBS-T buffer. Bands were detected using LAS imaging software (Fuji, New York, NY, USA).

Frozen tissue was homogenized in six volumes of the weight in homogenization buffer [50 mM Tris pH7.5, 300 mM NaCl, 5 mM EDTA, 1% Triton, 1x protease inhibitor (Sigma, catalog # 8340), 1x phosphatase inhibitor #1 (Sigma, catalog # P0044), 1x phosphatase inhibitor #2 (Sigma, catalog # P5726)], which was then stored in aliquots at −80 °C. An aliquot was further diluted to ten volumes of homogenization buffer. The samples were sonicated, and SDS was added to a final concentration of 2%. The samples were centrifuged for 20 min at 4 °C at 40,000 rpm. The supernatant was collected and protein concentration was determined using BCA protein assay (Pierce, Rockford, IL).

Briefly, cells were lysed using MPER lysis buffer (Thermo #78501) supplemented with 5% protease (Sigma #P8340) and 1% phosphatase inhibitors (Phosphatase Inhibitor 2 -Sigma P5726 and Phosphatase Inhibitor 3—Sigma P0044). Between 25–50 μg of protein for each sample were separated by electrophoresis through an 8% SDS-polyacrylamide gel and transferred to a PVDF membrane (Immobilon). Blots were blocked in 5% milk, 1% bovine serum albumin (BSA), tris-buffered saline and tween 20 (TBST) for 1 hour at room temperature. Blots were incubated overnight at 4°C and probed with the following primary antibodies: MARCKS (1:1000, Abcam, Ab52616), actin (1:1000, Santa Cruz, sc-1616), V-5 (1:5000, Invitrogen, R96125), lamin A/C (1:1000, Santa Cruz, sc-6215), α-Tubulin (1:2000, Santa Cruz, sc-53646). After overnight incubation, blots were washed 3 x 5 min washes in TBST. Approximately 1:5000 dilution of the appropriate secondary antibodies in 5% milk, 1% BSA, TBST were used and blots were incubated for 1 hour at room temperature. Bands were detected via a chemiluminescence approach as described previously and following PerkinElmer’s instructions (Western Lightning Plus ECL, NEL102001EA) [1 (link)].

Nuclei or cell pellets were lysed in RIPA buffer (pH 7.4) containing 100 mM NaF, 50 mM Hepes, 150 mM NaCl, 10% Glycerol, 1.2% Triton X, 1 mM MgCl₂, 1 mM EDTA, 1 mM Na₃VO₄, protease inhibitor cocktail (P8340; Sigma), phosphatase inhibitor 2 (P5726; Sigma), and phosphatase inhibitor 3 (P0044; Sigma). The supernatants collected after the first and second centrifuge steps of nuclear isolation were used after adding protease and phosphatase inhibitors. Total protein concentration was determined by Pierce BCA Protein Assay Kit (23225; Thermo). Lysates (50 μg protein) were run in 8% SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blocked for 10 min at room temperature with 5% milk and were incubated overnight at 4 °C with antibodies against Lamin A/C (1:1000, 4777; Cell Signaling Technology) or GAPDH (1:1000, 5174; Cell Signaling Technology). After three washes (10 min), the membranes were incubated for 1 h at RT with antibodies against rabbit IgG-HRP conjugate (1:1000, 170-6515: Bio-Rad) or mouse IgG-HRP conjugate (1:1000, 170-6516, Bio-Rad). After three 10-min washes, signals were visualized via Pierce ECL Western blotting substrate (PI32106; Thermo).

POT, DMSO7, GEF1 and TRM4 cell lines were tyripsinied and counted. Following seeding of 300,000 cells per well in six-well plates and incubation in a 37 °C and 5% CO₂ cell culture incubator overnight, three biological replicates of each cell lines were treated with DMSO, 40 nM of gefitinib and 100 nM of trametinib for 1 h. After the incubation under those conditions, cells were tyripsinised and centrifuged at 1500 rpm to generate cell pellets. Cell pellets were lysed using MDS Tris Lysis Buffer (Meso Scale Diagnostics) containing phosphatase inhibitor I (Sigma-Aldrich), phosphatase inhibitor II (Sigma-Aldrich), protease inhibitor (Cell Signalling Technology). Protein content of lysed samples was quantified using BCA assay (Sigma-Aldrich). MILLIPLEX MAP Akt/mTOR phosphoprotein kit, MILLIPLEX MAPK/SAPK signalling kit, MILLIPLEX MAP RTK phosphoprotein kit (48-611MAG, 48-660MAG, HPRTKMAG-01K respectively, MerckMillipore) were combined with the following singleplex magnetic bead sets to produce three multiplex Luminex assays; Total HSP27, GAPDH (46-702MAG, 46-710MAG, 46-623MAG, 46-641MAG, 46-608MAG, 46-667Mag, MerckMilipore). Bio-Plex Pro phosphor-PDGFRb and Akt (Thr308) (171-V50018M, 171-V50002, Bio-Rad) were combined into a triplex assay. Manufacturer’s recommendations were followed. Phosphoprotein levels were measured on the Luminex 200 system utilising xPOTENT c3.1 software.