Hrp linked anti rabbit igg secondary antibody

Mycelia were grown in the specified conditions and ground to a fine powder under liquid N₂ before being mixed with extraction buffer [50 mM Tris–HCl pH 7.0, 50 mM NaF, 1 mM NaVO₃, 1 mM DTT, phosphatase inhibitor cocktail P0044 (Sigma) and the complete mini EDTA-free protease inhibitor cocktail (Roche)]. Samples were centrifuged for 5 min at 14000× g and the protein concentration in the supernatant was determined as described above. 50 μg of total protein were run on pre-made gels, before being transferred to a membrane as described previously10 (link). Membrane blocking and washes, primary and secondary antibody incubation as well as membrane signal detection were carried out as described previously10 (link). The primary rabbit polyclonal IgG antibody anti-GFP (Abcam #ab290) was used in a 1:10000 dilution whereas a 1:5000 dilution was used for the secondary anti-rabbit IgG HRP linked antibody (Cell Signaling Technology, Beverly).

The epididymal fat pad was homogenized in an ice-cold protein lysis buffer containing 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM NaF, 1 mM Na₄P₂O₇, 1 mM β-glycerophosphate, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, and protease inhibitor cocktail (Complete Mini Protease Inhibitor Cocktail; Roche Diagnostics GmbH, Penzberg, Germany). The supernatant was obtained by centrifugation at 10,000× g for 30 min at 4 °C. A Bradford assay (Bio-Rad, Hercules, CA, USA) was used to determine the total protein concentration. Fifty micrograms of the protein sample was separated in 10% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (pH 7.6, TBST) for 1 h. The membrane was incubated with rabbit anti-mouse SIRT3 antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA) in 3% BSA/TBST overnight at 4 °C. The membrane was washed with TBST and then incubated with secondary anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Beverly, MA, USA) in 3% BSA/TBST for 1 h at room temperature. The signal was visualized by a chemiluminescent detection system and quantified using Quantity One analysis software (Bio-Rad, Hercules, CA, USA).

Proteins were isolated from snap-frozen thoracic aortic tissue of 2 heterozygous and 2 wild-type mice for each time point. The lysis buffer (RIPA, Sigma-Aldrich) was complemented with protease (Roche) and phosphatase inhibitors (Sigma-Aldrich). Protein samples were reduced by boiling and adding dithiothreitol (DTT) and loaded on a NuPage 4–12% Bis-Tris gel (Invitrogen) together with a 5× non reducing lane marker sample buffer (Thermo Scientific). Following SDS-PAGE, the proteins were transferred onto a nitrocellulose membrane using the iBlot dry blotting system (Invitrogen). The membranes were blocked in 2% ECL advantage blocking buffer (GE Healthcare) and incubated overnight at 4°C with primary antibody directed against phosphorylated p44/42 MAPK (ERK1/2 XP^TM rabbit mAb (Cell Signaling Technologies) (1/1000)). Subsequently, the membranes were incubated with secondary anti-rabbit IgG HRP-linked antibody (Cell Signaling Technologies) (1/5000). Membranes were developed with the SuperSignal West Dura chemiluminescent substrate (Pierce). Membranes were then stripped and re-blocked, in order to incubate with a primary antibody directed against the non-phosphorylated form of p44/42 MAPK (ERK1/2) (Cell Signaling Technologies). Next, the membranes were again incubated with the secondary antibody and developed. Quantification of the signal was performed using Image J software (NIH).

A 2× Laemmli sample buffer (BIO-RAD, CA, USA) containing 65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, and 0.01% bromophenol blue was used to extract protein samples which were then loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel (BIO-RAD, CA, USA). Electrophoresis was performed and proteins were then transferred onto nitrocellulose membranes. The membranes were blocked using 5% skim milk (Sigma-Aldrich, MO, USA) in TBS (Sigma-Aldrich, MO, USA) containing 0.05% Tween-20 (Sigma-Aldrich, MO, USA) for 1 h at room temperature, after which they were incubated overnight at 4 °C with the specific primary antibodies. The anti-GAPDH antibody (Millipore, Temecula, CA) was used to ensure equal protein loading. After incubation, the membranes were washed and incubated with HRP-conjugated secondary antibodies. Chemiluminescence was used to detect the protein bands. Antibodies used in this study included rabbit monoclonal anti-Bcl2 (Cell signaling, MA, USA), anti-Bcl-X_L (Cell signaling, MA, USA), rabbit polyclonal anti-Bax (Cell signaling, MA, USA), secondary anti-rabbit IgG HRP-linked antibodies (Cell signaling, MA, USA) and secondary anti-mouse IgG, HRP-linked antibodies (Cell signaling, MA, USA).

The MDA-MB-231 human epithelial breast cancer cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). The Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin and the antibiotic–antimycotic solution were purchased from GIBCO Life Technologies (Burlington, ON, Canada). Akt (#9272) (1:1000 dilution), p-Akt (Ser473) (#9271) (1:1000 dilution), mTOR (#2972) (1:1000 dilution), p-mTOR (Ser2448) (#2971) (1:1000 dilution), PARP (#9542) (1:1000 dilution), β-actin (#8457) (1:1000 dilution), as well as secondary anti-rabbit IgG HRP-linked antibodies (#7074) (1:2000 dilution) were from Cell Signaling Technology via New England Biolabs (Mississauga, ON, Canada). Bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Paclitaxel, and Metformin were from Sigma (Oakville, ON, Canada). Clarity Western enhanced chemiluminescence substrate (ECL), 30% acrylamide/bis solution 37 (5:1), ammonium persulfate (APS), polyvinylidene difluoride (PVDF) membranes and reagents for electrophoresis were purchased from Bio-Rad (Hercules, CA, USA).