Anti phospho s6 ribosomal protein

Equal amounts of protein from cell lysates were subjected to SDS-PAGE and transferred to a PVDF membrane (Merck-Millipore, Darmstadt, Germany). Where indicated, protein levels were quantified by densitometric analysis using the software ImageJ.^{43 (link)} The following antibodies were used: anti-TRAP1 (sc-13557), anti-β-actin (sc-69879) and anti-p70S6K (sc-230), from Santa Cruz Biotechnology; anti-phospho p70 S6 kinase (Thr389) (#9205), anti-E-Cadherin (#3195) and anti-phospho-S6 ribosomal protein (#2215) from Cell Signaling Technology (Danvers, MA, USA).

The antibodies used included mouse anti-FLCN mAb (N-terminal peptide: PQGDGNEDSPGQGEQC, produced at the Van Andel Research Institute), anti-Phospho-AKT Rabbit mAb (Cell Signaling), anti-Phospho-mTOR Rabbit mAb (Cell Signaling), anti-Phospho-S6 Ribosomal Protein (Cell Signaling). AQP1 (sc-25287, Santa Cruz Biotechnology); PAX2 (2549-1, Epitomics); CK7 (ab9021, Abcam; sc-80021, Santa Cruz Biotechnology) to determine RCC subtypes. Immunohistochemical analysis was performed following the manufacturers' protocols.

Proteins were extracted from frozen tumors using RIPA buffer (50 mM Tris–HCl (pH 8), 150 mM NaCl, 0.5% deoxycholic acid, 0.5% triton) supplemented with protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE and then electrophoretically transferred into nitrocellulose membrane and probed using the following primary antibodies: anti-GAPDH (V18 clone, 1/20000) purchased from Santa Cruz Biotechnology, anti-COX-2 (12282, 1/1000), anti-phospho Serin 473-AKT (4060, 1/2000), anti-PTEN (9552, 1/2000), anti-INPP4B (14543, 1/2000) and anti-phospho-S6 ribosomal protein (2211, 1/8000) purchased from Cell Signaling Technology (Ozyme). Proteins were detected according to the ECL Western Blotting Analysis System procedure (GE Healthcare, Buckinghamshire, UK). The intensity of the protein bands was quantified using ImageJ software.

The cells from the different growth conditions were taken and frozen to be broken afterward with one volume of glass beads in lysis buffer (Tris-HCl 0.1 M, pH 7.5, NaCl 0.5 M, MgCl₂ 0.1 M, NP40 1% (v/v), PMSF 10 mM and protease inhibitors and phosphatase inhibitors) in a FastPrep 24 shaker for three cycles of 20 seconds^{16 (link)}. Extracts were clarified by centrifugation and were diluted after quantification by the Bradford method (Biorad Inc. Hercules, CA, USA) in loading buffer for SDS-PAGE. After electrophoresis in an Invitrogen mini-gel device, the gel was blotted onto PVDF membranes for the Western blot analysis with a Novex semy dry blotter (Invitrogen, Carlsbad, CA, USA). The anti-phospho-S6 ribosomal protein (1:1000 dilution) and the anti-PRX antibodies (1:1000 dilution) were obtained from Cell Signalling Technology (Beverly, MA, USA). The anti-Rps6 antibody (1:1000 dilution) was acquired from Abcam (Cambridge, MA, USA), and anti-GFP (1:1000 dilution) and anti-HA (1:2000 dilution) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies HRP-conjugated were purchased from Santa Cruz Biotechnology and used at a dilution of 1:5000. The ECL Western blotting detection system (GE) was used following the manufacturer’s instructions.

Cells were grown to log phase (OD₆₀₀ of 0.8–1.2) in EMM supplemented with 225 mg l⁻¹ of uracil, arginine, histidine, adenine, leucine and then washed and transferred to EMM plus either low (45 mg l⁻¹) amino acids or high (1125, mg l⁻¹) amino acids. After 90 min of incubation, samples were taken and subjected to trichloroacetic acid (TCA) protein extraction. S. pombe cultures (5 ml) at an OD₆₀₀ of 0.8–1.2 were pelleted just after the addition of 100% TCA and washed in 20% TCA. The pellets were lysed with three 45-s pulses in a MAgNA lyser following the addition of glass beads and 100 μl 12.5% TCA. Cell lysates were pelleted for 20 min at 13,200 r.p.m., washed in acetone, and dried at 37 °C for 15 min. Pellets were resuspended in 50 μl of a solution containing 1% SDS, 100 mM Tris-HCl (pH 8.0), and 1 mM EDTA. For western blotting, proteins were separated on a 12% SDS–polyacrylamide gel electrophoresis gel, transferred onto a nitrocellulose filter (Amersham), and probed with anti Phospho-S6 Ribosomal Protein (1:500; Ser235/Ser236; #2211) and Phospho p70 S6 Kinase (1:500; Thr389; #9205, Cell Signaling Technology, Danvers, MA) primary antibodies. Actin was used as a loading control (MS1295P Thermo).