Cells were lysed in RIPA buffer (Thermo Fisher Scientific, Inc.), and the proteins contents were quantified using the BCA protein assay kit (Thermo Fisher Scientific, Inc.). 30 µg of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto nitrocellulose membranes (Schleicher & Schuell). The membranes were incubated in succession with the blocking buffer (5% BSA in PBS) for 2 h at room temperature, with the primary mouse monoclonal antibody anti-PIM1 diluted 1:250 (cat. no. ab54503), rabbit monoclonal antibodies anti-CDK4 diluted 1:1,000 (cat. no. ab108355), anti-cyclin D1 dilute 1:200 (cat. no. ab16663) or anti-cyclin E1 diluted 1:1,000 (cat. no. ab33911) (all from Abcam) overnight at 4°C and finally with horseradish-peroxidase conjugated goat anti-mouse diluted 1:200,000 (cat. no. STAR207P; Bio-Rad Laboratories, Inc.) or anti-rabbit IgG secondary antibodies diluted 1:200,000 (cat. no. STAR208P, Bio-Rad Laboratories, Inc.), incubated 2 h at room temperature. Protein blots were detected using an ECL Chemiluminescent Substrate (Cyanogen) and the intensity of the bands was quantitatively analysed using the Fluor-Sä MultiImager (Bio-Rad Laboratories, Inc.).
Star207p
Manufactured by Bio-Rad
The STAR207P is a laboratory instrument designed for automated liquid handling. It features a precise pipetting system and is capable of performing various liquid transfer operations with high accuracy and consistency.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Star208p, by Bio-Rad (1 mentions)
Thrombin, by Thermo Fisher Scientific (1 mentions)
Star124p, by Bio-Rad (1 mentions)
Glutathione resin, by GenScript (1 mentions)
Ab50010, by Abcam (1 mentions)
Ab75639, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
3 protocols using star207p
1
Protein Expression Analysis by Western Blotting
2
Recombinant Protein Purification and Antibody Generation
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The N-001 was prepared as described previously24 (link),25 (link). In brief, plasmid encoded constructs for C2I and C2II-C1 were expressed in E. coli BL21. E. coli was cultured in LB medium supplemented with ampicillin (100 µg/mL) at 37 °C and were induced at an optical density of ~ 0.6 at 600 nm wavelength with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). A French pressure cell was used to lyse the cell paste at 690 bar. Glutathione resin (Genscript) was used for affinity purification. GST fusion tags were removed using thrombin (Thermo Fisher). C2II-C1 was further activated using trypsin by incubation at 37 °C for 30 min at a 1:5 enzyme to substrate ratio as previously described45 (link). A custom-made polyclonal antibody targeting the N-001 enzymatic component C2I, was prepared in rabbits. The other antibodies used were goat anti-mouse IgG (H/L) polyclonal antibody (BioRad, Cat no. STAR207P) and goat anti-rabbit IgG (H/L): HRP (BioRad, Cat no. STAR124P). Thermo Scientific Pierce TMB substrate was used for ELISAs.
3
Quantifying Cardiac Nitric Oxide Synthases
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The tissue expression of iNOS, eNOS, and phosphorylated eNOS (phospho S1177) was determined using western blot. The aliquots of 60 µg of total protein from heart homogenates were separated on 12% SDS-PAGE. Then, iNOS, eNOS, and phospho-eNOS proteins were transferred on PVDF membranes (Bio-Rad) and were detected with primary antibody (mouse anti-iNOS polyclonal antibody 1 : 5000 (Abcam, ab21775); anti-eNOS (Abcam, ab50010); anti-phospho (S1177)-eNOS (Abcam, ab75639), respectively) and secondary goat anti-mouse IgG horseradish peroxidase conjugate 1 : 1000 (Bio-Rad, STAR207P). ClarityTM Western ECL (Bio-Rad) substrate was used for proteins detection. ChemiDocTM MP System and Quantity One Software (Bio-Rad) were used for detection of bands and measurement of their density. iNOS, eNOS, and phospho-eNOS quantities were expressed as AU normalized to total protein amount.
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