Trans blot transfer medium

Tissues and cells were lysed on ice for 30 min in RIPA buffer (100 mM Tris, 150 mM NaCl, 1% Triton, 1% deoxycholic acid, 0.1% SDS, 1 mM EDTA, and 2 mM NaF) supplemented with 1 mM sodium vanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, and 2 mM pepstatin A. The supernatant was collected after centrifugation at 12,000 rpm/min for 15 min, and protein concentration was determined using protein assay reagent from Bio-Rad (Hercules, CA, USA). Proteins were electrophoretically separated by SDS-PAGE and transferred to nitrocellulose (Trans-Blot Transfer Medium, Bio-Rad). Membranes were blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20, and incubated with specific antibodies. Protein bands were detected by incubation with horse radish peroxidase-conjugated antibodies (Cell signaling), and visualized through enhanced chemiluminescence reagent (Thermo Fisher).

ASCs and AMCs kept under experimental conditions (72 h and 21 days TNFα 300 U/mL) were scraped and incubated in ice for 30 min with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P40) and a protease inhibitor cocktail (Complete EDTA-free, Roche Molecular Biochemicals, Merck KGaA, Darmstadt, Germany). The total cellular lysate was centrifuged at 14,000 rpm for 1 h to clear cell debris. The protein concentration was determined using the Bradford assay. Proteins were denatured in Laemmli sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 62.5 mM Tris-HCl pH 6.8, 0.004% bromophenol blue), separated on 12% polyacrylamide gels, transferred to nitrocellulose membranes (TransBlot Transfer Medium Bio-Rad Laboratories S.r.l., Segrate, MI, Italy), and blotted with the primary antibodies listed in Table 2. Antigen–antibody complexes were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) on a CCD camera (Chemidoc, Bio-Rad Laboratories S.r.l., Segrate, MI, Italy).
The ASC52telo (SCRC-4000, ATCC, Manassas, VA, USA) hTERT immortalized adipose derived mesenchymal stem cell line was used as a positive control. Western blot bands were quantified by densitometry using ImageJ software V1.52 and the results were presented as histograms using GraphPad Prism 5 software (California).

Overnight cultures of cells transformed with plasmids encoding Rof variants were grown in LB + carbenicillin (100 mg/l) at 32 °C. After 1:100 dilution into fresh media, the cultures were grown to an early exponential phase at 32 °C, induced with 1 mM IPTG for 60 min, pelleted and frozen. Cell pellets were resuspended in PBS, sonicated and centrifuged. Protein concentration in cleared lysates was measured with Bradford reagent and normalized before resolving in SurePAGE Bis-Tris 8-16% gels (GenScript). Molecular weight markers were run on every gel to monitor separation. Proteins were transferred to a nitrocellulose membrane (Bio-Rad Trans-Blot Transfer Medium, Cat. 162-0112). Membrane was incubated with primary anti-HA antibodies from mice (1:5,000 dilution; Sigma-Aldrich, Cat. H9658) and with secondary anti-mouse horseradish peroxidase linked antibodies (1:10,000 dilution; Amersham, Cat. NA931V) before imaging using Bio-Rad Clarity Max Western ECL Substrate (Cat. 1705062 S) with a Bio-Rad ChemiDoc XRS+ system controlled using ImageLab (version 6.1; Bio-Rad).

Equal amounts of proteins were resolved in either 8 or 10% SDS-PAGE and electroblotted onto polyvinylidine difluoride membrane (PVDF) using semi-dry blotting system (Trans-Blot Transfer medium; Bio-Rad Laboratories). Membranes were blocked in 5% BSA in PBS containing 0.1% Tween-20 and incubated overnight at 4°C with antibodies against rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:1000), rabbit anti-ERK1/2 (1:1000), rabbit anti-phospho-AKT (Ser473) (1:500), rabbit anti-AKT (1:1000) from Cell Signaling Technology (Danvers, MA); mouse anti-EGFR (1:200) and anti-pan actin (1:50 000) antibodies from Neomarker (Fremont, CA). After several washes, membrane was incubated with either goat anti-rabbit or goat anti-mouse horseradish peroxidase conjugated secondary antibodies (DakoCytomation, Denmark) (1:10 000). Specific protein bands were visualized with an enhanced chemiluminescence using Western Lightning chemiluminescent kit (Perkin-Elmer, MA).

SDS–polyacrylamide gel electrophoresis was performed using 12% bis–tris gels (Biosharp), and proteins were transferred to PVDF membranes by semidry transfer using Trans-Blot transfer medium (Bio-Rad). Membranes were blocked in 5% skimmed milk in TBS-T and incubated overnight at 4 °C with primary antibodies, including γH2AX (#7918, Cell Signaling Technology), Caspase-3 (#14220, Cell Signaling Technology), Caspase-9(#9508, Cell Signaling Technology), Bax (#5023, Cell Signaling Technology), Bcl-2(#15071, Cell Signaling Technology), ER-a (#8644, Cell Signaling Technology), PR (#8757, Cell Signaling Technology), RAD51(14961-1-AP, Proteintech), Fas (ab133619, Abcam), FADD (ab124812, Abcam).

Protein lysates were obtained from about 50 mg of frozen samples of different brain areas, with the exception of centenarians, from which only frontal cortex samples were available. A lysis buffer with the following composition was used: CHAPS 4%, Urea 8 M, DTT 65 mM, Tris 40 mM, phosphatase, and protease inhibitors (Sigma). Lysis was performed with the OMNI TH Tissue Homogenizer (OMNI international). The lysate was then centrifuged at 25000 rpm for 1 h at 4°C and the supernatant was collected. Total protein extract was quantified by Bradford’s method and stored at −80° until the analysis; 50 μg of protein extract were separated on a 12% or 16% SDS-polyacrylamide gel, transferred to a Polyvinylidene Difluoride (PVDF) or nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad) and then immunoblotted with primary antibodies (Table 2). To evaluate the content of respiratory chain complexes and ATP synthase, blots of resolved proteins were incubated with primary mouse/rabbit monoclonal antibodies specific for single subunits of each OXPHOS complex as reported in Barbato et al. (2015) (link). As a loading control, GAPDH was used. Densitometry analysis of bands was performed using ImageJ software.