Non fat dried milk

Cell pellets were resuspended and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol), complemented with protease and phosphatase inhibitors cocktail (ThermoScientific). Total proteins extracts concentrations were determined through Bradford assay (Bio-Rad). Cytosol and nucleus protein fractions were obtained as previously described [47 (link)].
After 1 h blocking with 5% non-fat dried milk (EuroClone) or bovine serum albumin (SERVA) in Tris-buffered saline with 0.1% Tween (TBS-T), membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies used: anti-pFGFR1 (06-1433, Millipore), anti-FGFR1 (Abcam ab137765), anti-pSTAT3 (D3A7, Cell Signaling), anti-STAT3 (06596, Millipore), anti-pAKT1 (ab81283; Abcam), anti-AKT1 (ab32505; Abcam), anti-pERK1/2 (ab32538; Abcam), anti-ERK1/2 (ab17942; Abcam), anti t-GFP (TA150041, Origene) and β-actin (Sigma, A5441). After membrane incubation with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (Immuno Reagents), the positive bands were visualized using the ECL kit SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) as previously shown [48 (link)].

(3-Aminopropyl)triethoxysilane (APTES), bis(sulfosuccinimidyl)suberate (BS3), and H₂SO₄ were purchased from Sigma-Aldrich (MO, USA). Phosphate-buffered saline (PBS) was purchased from GIBCO (CA, USA). HCl was purchased from Romil (UK). Absolute ethanol and H₂O₂ were purchased from Carlo Erba (IT). Non-fat dried milk was purchased from EuroClone (IT). Protein A was purchased from Invitrogen (CA, USA). Mouse anti-His monoclonal antibody (AbaH) was purchased from Santa Cruz Biotechnology (CA, USA). Recombinant His-tagged p53 protein was kindly provided by Prof. Mariorosario Masullo.
Diatomite (Aulacoseira sp.) was a gift from Prof. Dusan Losic (University of Adelaide) and purified using the separation processes described elsewhere [13 (link)].
A simplified magnesiothermic reduction process, with respect to that firstly demonstrated by Sandhage group [9 (link)], was used to convert silicon dioxide of diatoms into pure silicon. We heated the magnesium (Mg) source and the diatom silica in a tungsten boat inside of a furnace to 650 °C under argon gas flow. After cooling, the process was repeated to ensure complete conversion. The Mg source and silica diatom frustules were thorough mixed without any other reducing agent in molar ratio of 1.25:1 Mg turnings to silica resulted in complete shape-preserving conversion. The reaction scheme is the following: $2\mathrm{M}{\mathrm{g}}_{\left(\mathrm{gas}\right)}+\mathrm{S}\mathrm{i}{{\mathrm{O}}_2}_{\left(\mathrm{solid}\right)}->2\mathrm{Mg}{\mathrm{O}}_{\left(\mathrm{gas}\right)}+\mathrm{S}{\mathrm{i}}_{\left(\mathrm{solid}\right)}$