Luminata forte

Whole cell lysates were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors. Proteins were resolved on 10–15% SDS-PAGE gels and electroblotted onto PVDF membrane. Membranes were blocked with 5% BSA or dried skimmed milk in TBS-T and incubated in primary antibodies in blocking buffer: anti-pRPA32-Ser³³ (A300-246A Bethyl Laboratories), anti-phospho-γH2AX-Ser¹³⁹ (Merck Millipore, 05–636), anti-pCHK1-Ser³⁴⁵ (Cell signalling #2348), anti-RNA polymerase II CTD repeat YSPTSPS (phospho-S5) (Abcam ab5401), anti-PUM2 (Bethyl Laboratories A300-202A), anti-RNASEH1 (SantaCruz Biotechnologies, H-4, sc-376,326), anti-β-actin (Abcam ab6276), anti-Vinculin (Merck Millipore, V9131), anti-GAPDH (Cell Signalling 14C10 #2118). Membranes were washed and incubated with HRP-conjugated secondary antibodies and blots developed with Luminata™ forte (Merck-Millipore) and imaged using iBright (Invitrogen).

After harvesting the cells by trypsin, the suspension was centrifuged (10,000 rpm, 10 min). Subsequent lysis of the pellet, separation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis, electroblotting on poly(vinylidene fluoride) membrane and blocking of non-specific binding was performed according to our previously published study^{46 (link)}. For experiments, primary anti-MT3 antibody (ab214314; dilution 1:750, Abcam, Cambridge, UK), anti-GFP antibody (ab183734, dilution 1:2,000, Abcam), anti-GAPDH (ab181602, dilution 1:5,000, Abcam) and anti-α-tubulin (ab7291, dilution 1:5,000, Abcam) were utilized. For development of the bands and their visualization using Azure c600 (Azure Biosystems, Dublin, CA, USA), we used Luminata Forte (EMD Millipore, Burlington, MA, USA). After visualization, western blots densitometric quantitation of signals was performed using ImageJ (v1.53 g, National Institute of Health, Bethesda, MA, USA).

MC3T3-E1 was treated with ALA and SFC for 1 h and then treated with LPS for 24 h. Brefeldin (3.0 μg/ml) were used to inhibit protein transport during culture. Cells were washed with PBS and treated with cell lysis buffer (5 mM EDTA, 10％ glycerol, 1％ Triton X-100, 0.1% SDS, 1％ NP-40) in PBS. Protein concentration in each of the lysates was measured with Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA) and adjusted to be the same for each lysate. After mixing with sample buffer, it was heat denatured, were electrophoresed on a TGX Precast gel (Bio-Rad Laboratories). The proteins were transferred to a PVDF membrane, and blocked with PVDF Blocking Reagent (Toyobo Co. Ltd, Osaka, Japan). Membrane was then incubated with anti-IL6 antibody (1/2000 dilution; ProteinTech Group, Chicago, IL, USA). After washing 0.5% Tween-20 in PBS (PBS-T), the membrane was incubated with HRP-conjugated secondary antibody (Thermo Fishter Scientific, San Jose, CA). To confirm the amount of the loaded protein were equal, membrane was incubated with anti-β Actin antibody (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Chemiluminescence was produced using Luminata Forte (EMD Millipore Corporation, Billerica, MA) and detected with LumiCube (Liponics, Tokyo, Japan).

Cell lysates of primary bone marrow-derived mouse or human blood neutrophils were prepared by lysing the cells with lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 200 μM Na3VO4 and 1 mM PMSF in H₂O. Shortly before use, a protease inhibitor cocktail was added to the lysis buffer. Cells were lysed for 25 min on ice, with frequent vortexing. After the lysis, supernatants were collected following high-speed centrifugation (10 min, 17,949 × g, 4 °C). In all, 30–50 μg of protein was loaded on 12% SDS polyacrylamide gels (Expedeon, Lucerna-Chem). The samples were separated by electrophoresis under reducing conditions and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Merck Millipore). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline solution containing 0.1% Tween 20 (TBST) for 1 h, followed by incubation with monoclonal anti-OPA1 antibody (clone 18/OPA1, Cat # 612606, BD Biosciences, dilution 1:1000) in TBST and 5% non-fat dry milk at 4 °C overnight. Membranes were washed using TBST, then incubated with the corresponding HRP-conjugated secondary antibody (Cat # GENA931, Sigma-Aldrich, dilution 1:10,000) and visualized by chemiluminescence (ECL Western Blotting Analysis System; GE Healthcare or Luminata Forte; Merck Millipore).

E. coli-derived met-hBMP-2 (GenScript) and CHO-derived hBMP-2 (Infuse^®) were analyzed under reducing and non-reducing conditions (Soares et al. 2000 (link)). Coomassie Brilliant Blue G-250 was used for the staining. For Western blotting, the semi-dry transfer technique was utilized on a nitrocellulose membrane, with anti-hBMP-2 affinity-purified rabbit IgG (1:2000) (Biovision, Milpitas, CA, USA) and goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000). Protein visualization was performed with Luminata Forte (Merck, Burlington, MA, USA) on X-ray film (CL-Xposure™ Film, Thermo Scientific, Rockford, IL, USA).

Cytoplasmic cell fraction was collected by using cell lysis buffer (20 mM Tris-HCl (pH 8.0; Wako), 2 mM EDTA (Wako), 0.5% NP-40 (Wako), 1 μM pepstatin (Sigma), 1 μM leupeptin (Sigma), 2 mM sodium orthovanadate (Wako), 1 μM calpain inhibitor (Sigma), phosphatase inhibitor cocktail I/II (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma)). The protein contained amount of these fraction was evaluated using a bicinchoninic acid protein-assay kit (Wako). The extracts (40 μg of protein) were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinyl difluoride membranes (GE Healthcare, Buckinghamshire, UK). The membranes were reacted with the following antibodies: anti-Bcl-2, anti-Bcl-xL, anti-Survivin, anti-MDR1, anti-BCRP, anti-MRP1, anti-LRP1 (Santa Cruz Biotechnologies, CA, USA), anti-phospho-Src (Tyr527), anti-Src (Cell Signaling Technology, Beverly, MA), and anti-β-actin (Sigma) as an internal control. The membranes were reacted with horseradish peroxidase-coupled secondary antibodies (GE Healthcare) for 1 h at room temperature and proteins were assessed using a Luminata Forte (Merck Millipore, Nottingham, UK).

For western blot analyses cells were collected 0, 6, 8, 12, 16, and 24 h after treatment, respectively. The cells were taken up in lysis buffer and stored at −80°C as described before (Grabiec et al., 2019 (link)). Protein concentrations were determined using the method of Bradford (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Barrington IL, United States) (Bradford, 1976 (link)) and 10 μg protein was loaded onto a 12.5% (w/v) sodium dodecyl sulfate–polyacrylamide gel. After gel electrophoresis, the proteins were blotted onto nitrocellulose membranes (Protran 0.45 μm, Amersham, Freiburg, Germany) and non-specific protein binding sites were blocked for 30 min with roti block solution (Carl Roth, Karlsruhe, Germany). The nitrocellulose membranes were incubated with the antibody against IMPDH2 (Table 1) [diluted 1:2000 in roti block solution containing 0.2% (v/v) Tween 20] until the next day at 4°C. After incubation with horseradish peroxidase conjugated antibodies (Table 1) the chemiluminescent (Luminata Forte, Merck) signal was detected with Fusion X (VWR, Radnor, PA, United States). For semi quantitative analysis, the relative signal intensities of the immunoreactive bands were determined and the IMPDH2 intensity was normalized to the ß-actin (Table 1) intensity. Fusion FX7 with FusionCapt Advance Solo software (VWR) was used for analysis.