Clarity western ecl chemiluminescent substrate

Western blot analysis was performed as already described [32 (link), 40 (link)]. Briefly, for protein detection, primary antibodies anti-SGPL1 ((H-300) #sc-67368; Santa Cruz, USA), anti-SPHK1 ((M-209) #sc-48825; Santa Cruz, USA), anti-SPHK2 ((P-19) #sc-22704; Santa Cruz, USA), anti-PCNA ((PC10) #sc-56; Santa Cruz, USA), anti-β-Actin ((C4) #sc-47778; Santa Cruz, USA), anti-MTSS1 ((SS-3) #sc-101204; Santa Cruz, USA), anti-PARP1 ((B-10) #sc-74470; Santa Cruz, USA) and anti-Bcl-2 ((C-2) #sc-7382; Santa Cruz, USA) were incubated overnight at 4 °C followed by labeling with a horseradish peroxidase (HPR)-conjugated secondary antibody (mouse #7076; rabbit #7074P2; Cell Signaling, USA) for 1 h at room temperature. Finally, the protein signals were visualized with the Clarity™ Western ECL Chemiluminescent Substrate (Bio-Rad Laboratories Inc., USA). Stainfree-images and β-actin were used as loading control. Band intensity was analyzed densitometrically with the Molecular Imager ChemiDoc XRS and Image Lab 3.0.1 software (Bio-Rad, München, Germany). Protein detection was repeated at least three times with individually prepared cell lysates from independent passaged cells.

hMADS cell lysates were obtained by using lysis buffer containing 50 mM Tris–HCl (pH 7.4), 1% NP-40, 1 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonylfluoride, and 50 mg/ml aprotinin. Samples were centrifuged, and protein concentrations were determined by the Bradford Protein Assay (Bio-Rad Laboratories, Segrate, Italy). Proteins were separated by SDS-PAGE then transferred to a nitrocellulose membrane using the Trans-Blot Turbo^TM Transfer system (Bio-Rad). To check loading and transfer efficiency, membranes were visualized with Ponceau S solution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were then blocked for 1 h at room temperature (RT) in TBS-Tween-20 (50 mM Tris-HCL [pH 7.6], 200 mM NaCl and 0.1% Tween-20) containing 5% non-fat dried milk and subsequently incubated overnight at 4°C with the primary antibody (Table 1B). After washing in TBS-Tween-20 and incubation with the secondary antibody for 1 h at RT (Table 1C), bands were visualized with the Chemidoc Imaging system using the Clarity™ Western ECL chemiluminescent substrate (all from Bio-Rad). Quantitation of immunoreactive bands was performed using the Bio-Rad Image Lab software. Where appropriate, membranes were stripped, washed and re-probed for total protein content.

Liver tissue lysates were obtained by homogenisation in RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X‐100, 5 mM EDTA, pH 8.0) containing a protease inhibitor cocktail (Roche Applied Science). Samples were centrifuged and protein concentrations were determined by the Bradford assay (Bio‐Rad Laboratories). Total protein extracts (20–15 μg) were separated by 10% SDS‐PAGE and transferred to a nitrocellulose membrane using Bio‐Rad's Trans‐Blot Turbo TM Transfer system. Loading and transfer efficiency were checked by Ponceau staining (Santa Cruz Biotechnology). Membranes were then blocked for 1 h at room temperature (RT) in TBS‐Tween 20 containing 5% non‐fat dried milk and subsequently incubated overnight with primary antibodies (Table 1a). Finally, they were incubated with a secondary antibody conjugated to horseradish peroxidase (Vector Laboratories; Table 1b) for 1 h at RT. Bands were visualised with the Chemidoc Imaging System using the Clarity™ Western ECL chemiluminescent substrate (all from Bio‐Rad). Densitometric analysis was performed with Bio‐Rad's Image Lab software.

Expression of SOD and catalase was determined by the SDS-PAGE and semiquantitative western blot analysis. Samples of erythrocyte lysates (20 μg protein) were separated by SDS-PAGE on 6–15% gradient polyacrylamide gels for determination of SOD1 and catalase protein levels. Separated proteins were transferred to PVDF membranes (Millipore Corp., USA) using semidry Trans-Blot Turbo Blotting System (BioRad, USA). The membranes were probed with primary antibodies specific for SOD1 (1 : 500, Santa Cruz Biotechnology, USA) and catalase (1 : 2000, Millipore Corp., USA). A peroxidase-linked antirabbit IgG (1 : 10 000, Santa Cruz Biotechnology, USA) was used as the secondary antibody. Immunoreactive proteins were visualised by Clarity Western ECL Chemiluminescent Substrate (BioRad, USA), checked for the protein load and band densities, and analyzed on ChemiDoc MP imaging system (BioRad, USA).

Western blot analysis was performed as already described [21 (link), 30 (link), 34 (link)]. Briefly, for protein detection, primary antibodies anti-β-Actin ((C4) #sc-47,778; Santa Cruz, Dallas, USA), anti-PCNA ((PC10) #sc-56; Santa Cruz, Dallas, USA), anti-AMT (#10633–1-AP; Proteintech Europe, Manchester, UK), anti-GCSH (#16726–1-AP; Proteintech Europe, Manchester, UK), anti-SGPL1 ((H-300) #sc-67,368; Santa Cruz, Dallas, USA), anti-Ezrin ((3C12) #sc-58,758; Santa Cruz, Dallas, USA), anti-CXCR4 (#11073–2-AP; Proteintech Europe, Manchester, UK) P-Cadherin (#13773–1-AP; Proteintech Europe, Manchester, UK) and Stathmin (#3352; Cell Signaling, Danvers, USA) were incubated overnight at 4 °C followed by labelling with a horseradish peroxidase (HPR)-conjugated secondary antibody (mouse #7076; rabbit #7074P2; Cell Signaling, Danvers, USA) for 1 h at room temperature. Finally, the protein signals were visualized with the Clarity™ Western ECL Chemiluminescent Substrate (Bio-Rad Laboratories Inc., USA). Stain free-images and β-actin were used as loading control. Band intensity was analyzed densitometrically with the Molecular Imager ChemiDoc XRS and Image Lab 6.0.1 software (Bio-Rad, München, Germany). Protein detection was repeated at least three times with individually prepared cell lysates from independent passaged cells.

Cells were harvested and homogenized in the RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM MgCl₂, 1.5% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate) supplemented with protease cocktail inhibitor, 10 μM MG-132, 10 μg ml⁻¹ leupeptin and 1 mM phenylmethylsulfonyl fluoride. The protein concentrations of lysates were determined using BCA method (Beyotime). Samples were mixed with the membrane protein solubilization buffer (62.5 mM Tris-HCl, pH 6.8, 15% SDS, 8 M urea, 10% glycerol, and 100 mM DTT) plus the 4× loading buffer (150 mM Tris-HCl, pH 6.8, 12% SDS, 30% glycerol, 6% 2-mercaptoethanol, and 0.02% bromophenol blue) and incubated for 30 min at 37 °C. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare). Immnoblots were blocked with 5% non-fat milk in TBS containing 0.075% Tween-20 (TBST) and probed with indicated antibodies overnight at 4 °C. After washing in TBST for five times, blots were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Immunoreactivity was developed with Clarity Western ECL chemiluminescent substrates (170–5061, Bio-Rad). Western blot images were captured by Amersham Imager 600 (GE Healthcare), and integrated optical intensities of each band were quantified by Image-Pro Plus 6 software (Media Cybernetics). Uncropped blots are shown in Supplementary Figs. 9–13.

Protein lysates were loaded and resolved under denaturing conditions on a NuPAGE 3% to 8% Tris-Acetate gel or NuPAGE 4% to 20% Tris-Glycine gel. Lysate was transferred to a PVDF membrane overnight at 40 mA at 4°C. Skim milk at 5% in 1× TBST buffer was used as a blocking agent. Primary antibodies used for Western blotting include mouse anti-FLAG 1:1000 (Sigma-Aldrich F1804), rabbit anti-HA 1:1000 (CST C29F4), rabbit anti-MYOM1 1:1000 (Abcam ab201228), mouse anti-cardiac troponin T 1:500 (Abcam ab8295), rabbit anti-MYOM2 1:500 (Abcam ab93915), rabbit anti-TUBB 1:1000 (Abcam ab6046), mouse anti-MuRF1 (muscle RING-finger protein 1) 1:1000 (Santa Cruz sc-398608), and rabbit anti-CAPN3 (calpain 3) 1:1000 (Proteintech 104-92-1-AP). Blots were blocked for at least 30 minutes at room temperature before addition to primary antibodies, incubated at 4°C overnight, and washed 3× with TBST before addition of mouse or rabbit secondary antibodies (Abcam ab97023, ab205718). Secondary antibodies were applied for 1 to 2 hours at 1:5000, washed 3× with TBST, and detected using Clarity Western ECL chemiluminescent substrates per the manufacturer’s protocol (Bio-Rad 1705060).