Versadoc mp 4000 system

Aliquots of the IPVL extracts (100 µg) in a final volume of 125 µL of rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS wt/vol, 40 mM DTT, and 2% pharmalyte 3-10 NL), were applied to 7 cm IPG strip (pH range 3 to 10 NL; GE Healthcare). Then, IEF and two-dimensional electrophoresis were conducted as described in our previous study [115 (link)]. After 2DE, hen IPVL proteins were transferred to a nitrocellulose (NC) membrane (0.2 µm, Sigma-Aldrich) in a Mini Trans-Biol Cell (Bio-Rad, Hercules, CA, USA), according to Słowińska et al. [116 (link)]. The NC membranes were incubated overnight at 4 °C with anti-ubiquitin antibodies (U5379, Sigma-Aldrich) diluted with TBS-T (0.05 M Tris-HCl, 0.15 M NaCl, and 0.1% Tween 20; pH 7.6) at a ratio of 1:100. Blots were scanned with a VersaDoc MP 4000 system (Bio-Rad). The proteins of interest were excised manually, according to the Western signal, and prepared for mass spectrometry identification according to the protocol developed by Luque-Garcia et al. [117 (link)]. Antibodies were removed by three washes of the protein spots with 20 mM sodium bicarbonate buffer (pH 7.4), followed by washing with 100 mM glycine (pH 2.4). On-membrane digestion and sample preparation for mass spectrometry were performed according to our previous study [16 (link)].

Total protein from cells or tissue samples was extracted using 1×RIPA buffer [42 (link)] containing protease inhibitor (P8340, Sigma, St. Louis, MO, USA). Then the lysate was incubated for 30 min on ice and centrifuged at 12,000 rpm for 15 min at 4 °C. The protein concentration was measured using Pierce BCA Protein Assay Kit (23225, Thermo Scientific, Waltham, PA, USA). Then, 70 µg protein from each sample was separated using SDS-polyacrylamide gel electrophoresis (10%, w/v) and blotted onto nitrocellulose membrane. The membranes were blocked in 5% (v/v) skimmed milk for 2 h, and then incubated overnight at 4 °C with diluted primary antibodies: FPN (sc-49668, Santa Cruz, 1:200), β-actin (AP0060, Bioworld, 1:10,000). After three times washes (10 min each time) in TBST with 0.1% Tween at room temperature, secondary antibodies were incubated for 2 h at room temperature. Following extensive washing with TBST, the ECL signal intensities were quantified using the VERSADOC MP 4000 system (Bio-Rad, California, CA, USA). Antibody against β-actin served as an endogenous reference.

Gels were scanned using the VersaDoc MP 4000 System (Bio-Rad Laboratories Inc., Hercules, CA, USA). PDQuest 8.0.1 Advanced software (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used for the analysis of the protein maps. The Spot Detection Wizard was used to select the parameters for spot detection, such as a faint spot and a large spot cluster. The results of automated spot detection were checked and manually corrected. A local regression model (Loess) was used for the normalization of spot intensity. The protein expression was accessed using PDQuest 8.0.1 Advanced software and was presented as the mean total intensity of a defined spot in a replicate gel group. The spot quantity is the sum of the intensities of pixels inside the boundary. The fold of the protein expression change was accessed based on the mean protein intensity. For quantitative differentiation, a 1.5-fold change or higher in the average spot intensity was regarded as significant. The statistical significance of differences was assessed using the student’s t-test at a significance level of 0.05 in three replicates.

Cells or tissue samples were lysed in RIPA lysis buffer (Tian P. et al., 2017 (link)) added protease inhibitor (P8340, Sigma, St. Louis, MO, United States) for 30 min on ice, and then centrifuged at 12, 000 rpm for 15 min at 4°C. The supernatant was collected and measured to calculate protein concentration using a BCA protein assay kit (23225, Thermo Fisher Scientific, Waltham, PA, United States). After denaturation, 80 μg protein was electrophoresed in a 10% SDS-PAGE, and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder in TBST (Tris buffer with 0.1% Tween, pH 7.6) for 2 h and then incubated at 4°C overnight with primary antibodies: DMT1 (ab55735, Abcam, 1:500) or β-actin (BS6007M, Bioworld, 1:10000). A secondary HRP-conjugated antibody (BS12478, Bioworld, 1:10000) was used to incubate the membrane for 2 h prior to chemiluminescent detection. The signals were determined using a chemiluminescent substrate (ECL) kit (NCI4106, Thermo Fisher Scientific, Waltham, PA, United States). Then, protein intensities were quantified by the VersaDoc MP 4000 system (Bio-Rad, California, CA, United States).

Proliferative signals were investigated using ERK1/2 phosphorylation, as previously described [31 (link), 32 (link)]. To these purposes, cell lines were seeded in 24-well plates (1.0x10⁵ cells/well) and treated 15 min with 1 μM 8-Br-cGMP or 50 μM vardenafil. Where needed, cells were pretreated 1 h with the NS 2028 GC inhibitor, PMA or U0126 [24 (link)]. Cells were lysed in ice-cold RIPA buffer containing protease and phosphatase inhibitors for protein extractions. The phosphorylation of ERK1/2 was evaluated using an anti-pERK1/2 primary antibody (#9101, Cell Signaling Technology Inc., Danvers, MA, USA). Lysed samples were loaded to separate proteins in a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The anti-total ERK antibody (#9102, Cell Signaling Technology Inc.) was used as a loading control. Then, membranes were incubated with a secondary anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (#NA9340V; GE HealthCare).
Western blotting signals were revealed upon incubation with ECL chemiluminescent compound (GE HealthCare, Chicago, IL, USA). Images were acquired by the VersaDoc™ MP 4000 System and QuantityOne analysis software (Bio-Rad Laboratories Inc., Hercules, CA, USA).

The SL construct’s target region was PCR-amplified using HotStarTaq Master Mix Kit (Qiagen). The PCR reaction was carried out under the following conditions: 95 °C for 15 min, 37 cycles of 94 °C for 40 sec, 54 °C for 1 min, and 72 °C for 1 min, and one cycle at 72 °C for 10 min. 5 µL of the PCR product was then digested with 0.5 µL Thermo Scientific FastDigest XhoI enzyme in a total reaction volume of 15 µL, followed by gel electrophoresis on a 2% agarose gel in TAE buffer. The gel was visualized on a Molecular Imager^® VersaDoc™ MP 4000 system, and the band’s intensities were quantified using Image Lab software (Bio-rad, Hercules, CA, USA). The percentage of indels was calculated as ((a/(a + b + c)) × 100), where a is a band percentage of the undigested PCR product and b and c are the band percentages of the digested product.