Fuji medical x ray film

Proteins were transferred from SDS‐PAGE gels onto nitrocellulose membranes (Amersham) in 1× standard transfer buffer at 15 V for 1 h. Membranes were blocked in 3% milk powder or 3% BSA in PBST for 1 h at room temperature or overnight at 4°C. Membranes were probed with primary antibodies in PBST, washed 3 times for 5 min in PBST and then probed with secondary antibodies. Membranes were washed 3 times for 2–5 min in PBS and incubated with Supersignal West Pro Plus Chemiluminescent Substrate for 5 min. For protein detection, films were exposed on Fuji Medical X‐Ray Films (Fuji) in exposure times ranged from 10 s to 20 min. Films were developed using a Film Processor (AFP Imaging). Film exposure and developing were conducted in the dark.

Cell lysates were run on a 12.5% polyacrylamide gel and transferred onto Amersham^™ Protan^™ 0.2 μm nitrocellulose blotting membranes (GE Healthcare). Membranes were stained using anti-ICAM1 (Santa Cruz Biotechnology), anti-actin (Sigma Aldrich), anti-VWF (Dako) overnight at 4°C and counterstained with anti-mouse HRP (Dako) or anti-rabbit HRP (Dako) for 1 hour. Immobilized antigens were detected by chemiluminescence using Pierce^®ECL Western Blotting Substrate (Thermo Scientific) and exposed on Fuji Medical X-Ray films (Fujifilm).

Western blot analyses were performed as previously described [2 (link)]. Briefly, 20 µg of total protein were loaded onto sodium dodecyl sulfate-polyacrylamide gels, electrophoresed, transferred to polyvinylidene difluoride membranes, blocked in 5% skim milk, and probed with the following antibodies: anti-AK2 [24 (link)], monoclonal anti-β-actin Clone AC-15 (Sigma), human integrin alpha M/CD11b (238439, R & D systems, Minneapolis, MN, USA) as primary antibodies, and anti-goat HRP IgG (Dako, Glostrup, Denmark), ECL anti-rabbit IgG horseradish peroxidase-linked whole antibody, and anti-mouse IgG (GE Healthcare, Munich, Germany) as second antibodies. Signals were detected using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) and Fuji medical X-ray films (Fujifilm, Tokyo, Japan) or ECL Prime Western Blotting Detection Reagent (Cytiva, Tokyo, Japan) and Omega Lum G (Aplegen, Inc., Pleasanton, CA, USA).

Whole-cell extracts were prepared using RIPA buffer containing proteinase inhibitors (Roche) and resolved using SDS-PAGE (Mini-PROTEAN TGX, Bio-Rad) under reducing conditions (β-mercaptoethanol, Bio-Rad) and Precision Plus Protein Standards (dual color, Bio-Rad) was used. Proteins were subsequently blotted onto a nitrocellulose membrane (Bio-Rad) and hybridized using anti-human antibodies against β-ACTIN (Sigma, 1:15,000), DNM1L (Santa Cruz Biotechnology, 1:200), MFN1 and MFN2 (Abcam, 1:1,000), PPARGC1A (Calbiochem, 1:1,000), and horseradish peroxidase–coupled secondary antibodies (1:6,000, Promega). The Pierce enhanced chemiluminescence (Thermo Fisher Scientific) detection system and Fuji Medical X-ray Films (FUJIFILM) was used. Quantification of densitometric units was performed using Scion Image (SCR_008673).

Cells were maintained in proliferation or differentiation media, washed with PBS and harvested by trypsination and pellet by centrifugation at 1500 rpm during 5 min at +4 °C. After one more wash step, a dry pellet was obtained and stored at −80 °C. To extract proteins, the pellet was dissolved in extraction buffer based on classic RIPA buffer (25 mM Tris-HCL, 50 mM NaCl, 0.5% deoxycholic acid, 2% Nonidet P-40, 0.2% SDS) supplemented with a protease inhibitors cocktail (cOmplete from Roche) and phosphatases inhibitors (PhosSTOP from Roche). The lysis was performed on ice during 30 minutes. After sonication, protein concentration was determined by a Lowry-like assay using the DC protein Assay from BioRad. Dosages were performed according to manufacturer’s specifications. An amount of 20 µg of total lysate was loaded onto 12% precasted MiniPROTEAN® TGX gels (Bio-Rad). Transfer was performed onto 0.22 µm PVDF membranes. After being blocked in 5% non-fat dried milk dissolved in TBS 1X, membranes were incubated overnight with primary antibodies (see Supplementary Table S4). After three wash steps with TBS, membranes were probed one hour with appropriate secondary antibodies. Following three new wash steps with TBS, membranes were developed using the ECL reagent (GE Healthcare) or ECL2 reagent (ThermoScientific) on Fuji Medical X-Ray Films (FujiFilm).

Liver homogenate containing 30 μg of tissue was subjected to protein denaturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [31 (link)]. The separated proteins were transferred onto polyvinylidene difluoride membranes through electroblotting for 1 h (100 V). After blocking with 5% nonfat milk in Tris-buffer saline, the membranes were incubated overnight in primary antibody at 4°C. Subsequently, the membranes were incubated in a secondary antibody at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit (Amersham) and Fuji medical X-ray films, and the relative densities were quantified.