Tbst buffer

In brief, cells were gathered in Radioimmunoprecipitation assay buffer (RIPA) (Thermo Scientific) supplemented with phosphatase–proteinase inhibitor (Thermo Scientific) and kept at 4 °C overnight. Eight micrograms of sample were run on NuPAGE 4–12% Bis-Tris gels (Invitrogen) in MOPS SDS Running Buffer (life technologies) and transferred to a nitrocellulose membrane using the Bio-Rad Trans-Blot Turbo Transfer System. Blots were blocked in 5% bovine albumin serum (BSA, Sigma-Aldrich) in TBS-T buffer (Bio-Rad) and subsequently incubated in primary antibodies in 5% BSA in Tris-buffered Saline (Bio-Rad) with 0.1% Triton X-100 (Sigma-Aldrich) (TBS-T buffer) at 4 °C overnight. The next day, blots were incubated in secondary antibodies in 5% BSA. Enhanced luminol-based chemiluminescent (ECL) detection was performed using the Pierce™ ECL Western Blotting Substrate (Thermo Scientific). Images were processed and analyzed using ImageJ/FIJI. The following primary antibodies were used: anti-TSC2 (rabbit, Cell Signaling, 1:500), anti-pS6 (rabbit, 1:2000, Cell Signaling), anti-S6 (rabbit, Cell Signaling, 1:8000), horseradish peroxidase (HRP)-conjugated anti-Actin (mouse, Abcam ab49900, 1:25,000), and HRP-conjugated anti-rabbit (goat, Abcam ab6721, 1:8000).

Cells were lysed with RIPA buffer (Thermo Scientific) containing a protease and phosphatase inhibitor mix (Sigma). The cell lysate was clarified, and the total protein of each sample was quantified using a BCA assay kit (Thermo Scientific). For PAGE, 10 μg of protein was mixed with LDS sample buffer and β-mercaptoethanol (Invitrogen). Samples were then heated for 5 min at 95 °C and briefly centrifuged before being loaded onto a Tris-glycine 4–20% gel in Tris-Gly running buffer (Nusep-Generon, Slough, UK). Proteins were transferred to a nitrocellulose membrane (Novex, ThermoFisher), which was then blocked with 5% milk powder (Bio-rad, Watford, UK) or 5% BSA in TBST buffer (Sigma) based on the antibody protocol. The membrane was then incubated with anti-RUNX2 (Bio-techne, Abingdon, UK), anti-osteopontin (Abcam, Cambridge, UK), and anti-GAPDH (Abcam) overnight at 4 °C under constant mixing. The membrane was washed with TBST 3× before incubation with anti-rabbit-HRP, anti-goat-HRP, or anti-mouse-HRP (1:1000) (Abcam) for 1 h at room temperature. The membrane was washed 5× in TBST, and chemiluminescence was developed using a SuperSignal WestPico PLUS chemiluminescent kit (Thermo Scientific) followed by image capture using an iBright 1500 imaging system. The image was analyzed using ImageJ, and the data obtained from RUNX2 and osteopontin was normalized to the GAPDH.

Prior to immunoblot analysis, cell lysates were diluted in 2X loading buffer (62.5 mm Tris–HCl (pH 6.8) (Sigma-Aldrich, USA), 20% glycerol (Invitrogen, CA, USA), 2% SDS (Invitrogen, NY, USA), 4% β-mercaptoethanol (Sigma-Aldrich, CA, USA), 0.008% bromophenol blue (Bio-rad, CA, USA)) and boiled at 100°C, 5 min. Proteins were then resolved via SDS-PAGE (BioRad, USA) and transferred onto nitrocellulose (GE Healthcare, USA) membranes at either 100 V for 2 h or 30 V for 16 h. The membranes were then blocked with 5% non-fat milk in TBS-T buffer (25-mm Tris–HCl, pH 7.4, 137-mm NaCl, 2.68-mm KCl and 0.05% Tween-20) (Sigma-Aldrich, USA) for at least 1 h at room temperature (RT). The membranes were then incubated with desired primary antibodies (Table 2) and rotated in 4°C overnight. Subsequently, the membranes were washed in TBS-T buffer and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Table 2) for 1–2 h at RT. HRP signals, which correspond to protein levels, were detected by various ECL detection reagents (ECL Western Blotting Substrate (Pierce, ThermoScientific, USA), Amersham ECL Western Blotting Detection Reagents (GE Healthcare, USA) and WesternBright Sirius Western Blotting detection kit (Advansta, CA, USA)).

Cells were lysed with RIPA buffer containing a protease and phosphatase inhibitor mix (Sigma). Cell lysate was clarified and the total protein of each sample was quantified using a BCA assay kit (Fisher). For PAGE, 30μg of protein was mixed with LDS sample buffer with added β-mercaptoethanol (Invitrogen). Samples were then heated for 10mins at 70°C and briefly centrifuged before being loaded onto a Tris-Glycine 4–20% gel in Tris-Gly running buffer (NuSep). Proteins were transferred to PVDF membrane (Fisher) which was then blocked with 5% milk powder (Co-operative) in TBST buffer (Sigma). The membrane was then incubated with anti-LRP6, anti (Ser1490) phospho-LRP6, anti-LRP5 (all New England Biolabs), anti-phospho-LRP5 (Abnova) or anti-GAPDH (Abcam) overnight at 4°C with constant mixing. The membrane was washed with TBST 3x before incubation with Anti-rabbit-HRP or anti-Mouse-HRP (1:1000) (Abcam) for 1h at room temp. The membrane was washed 5x in TBST and chemiluminescence developed using a PicoWest chemiluminescent kit (Thermo Pierce) followed by image capture using a Protein Simple Flourchem M imaging system.

Proteins samples were run on 4–20% Criterion TGX pre-cast gels (Biorad) in SDS/Tris-glycine running buffer and transferred to PVDF membranes by semi-dry trans-Blot Turbo system (Biorad). Membranes were blocked with either Odyssey Blocking Buffer (Licor Cat #927–40,000) or 5% skimmed milk in TBST buffer (SIGMA) and incubated for 1 h at room temperature or overnight at 4°. The membranes were washed with three 5 min wash cycles in TBST at room temperature (RT) followed by incubation for 1 h at RT with goat anti-mouse or rabbit IR Dye 680 or 800 antibodies (LICOR) or HRP-conjugated anti-mouse or rabbit secondary antibodies (Cambridge bioscience). Blots were washed as above at RT and scanned on an ODYSSEY® CLx (Licor). For HRP-secondaries, each membrane is soaked in SuperSignal® West Fempto/Pico maximum sensitivity substrate (equal parts Luminol and stable peroxide buffer, Thermo Scientific). Membranes were imaged using a GeneGenome XRQ Chemilluminescence imager (high quantium efficiency (QE) camera – Syngene). Quantitation of western blots was performed using Image Studio (Licor) or GeneSnap software.

Degrasyn (WP1130) and PR-619 were purchased from Selleck Chemicals (Houston, TX, USA). FBS, L-glutamine, penicillin and streptomycin solution, 0.25% trypsin EDTA, trypan blue, poly-L-lysine, ribonuclease A, propidium iodide (PI), RIPA buffer, SigmaFAST Protease Inhibitor Cocktail, and TBST buffer were acquired from Sigma-Aldrich (Steinheim, Germany). Annexin V-FITC was obtained from Immunostep (Salamanca, Spain). The CellEvent^® Caspase3/7 Green Flow Cytometry Assay by ThermoFisher (Waltham, MA, USA) was used. Anti-USP5 (sc-390943), anti-USP9X/Y (sc-365353), anti-USP14 (sc-515812), anti-Bcl-2 (sc-7382), and anti-β actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), whereas the anti-Bcl-xl (#2764) antibody was purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA). The anti-γH2A.X (ab26350) antibody was from Abcam (Cambridge, UK), and anti-mouse/HRP and anti-rabbit/HRP antibodies were bought from Dako (Glostrup, Denmark).