Readypreptm protein extraction kit

The proteins were separated from each sample by Ready Prep TM protein extraction kit (Bio Rad Inc, USA), by RIPA lysis buffer (Bio BASIC INC, Canada) with additional protease inhibitor and phosphatase inhibitor buffer. Then, the samples were centrifuged at ∼16,000×g for 30 minutes at 4°C to separate the supernatant and the amount of the sample was measured with the Bradford Assay Kit (Bio Basic Inc, Canada). After the separation of the antigen samples by sodium dodecyl sulfate-polyacrylamide, gel electrophoresis (SDS-PAGE) gel (Bio-Rad Inc, USA) to the immunoblotting membrane (PVDF) membrane blocked with 3% bovine serum albumin (BSA) using Bio-Rad Trans-Blot Turbo. The MDM2 and E-cadherin proteins were incubated with their diluted primary at 4°C overnight and washed with TBST and secondary antibodies conjugated with HRP enzymes. Then, chemiluminescent signals were measured and the bands were analyzed using ChemiDoc MP Imager against beta-actin [23 (link)].

The midbrain and the striatum tissue lysates were prepared in RIPA buffer. Protein extraction from supernatants was performed using a ReadyPrepTM protein extraction kit (Catalogue No. #163–2086, Bio-Rad Inc.). Protein was determined in samples using the Bradford Protein Assay Kit (Catalogue No. #SK3041, Markham, Ontario, Canada). Equal protein concentrations from all samples were loaded with sample buffer 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris HCl. After boiling for 5 min, samples were loaded on polyacrylamide gel (SDS-PAGE) (Catalogue No. #161–0181, Bio-Rad Inc). A sandwich of PVD and gel was transferred with transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol), allowing transfer from the gel to the membrane using the BioRad Trans-Blot Turbo apparatus. After membrane blocking for 1 h, incubation with primary antibodies was performed at 4 °C. Afterwards, the solution was added to the HRP-conjugated secondary antibody (Novus Biologicals, USA). Then, the ECL chemiluminescent substrate (Catalogue No.# 170–5060, Bio-Rad, Inc.) was added. The chemiluminescence was captured, and image analysis was performed against β-actin using a ChemiDoc MP imager.

The ReadyPrepTM protein extraction kit (total protein) from Bio-Rad Inc was employed according to manufacturer instructions and was added to each sample of the homogenized renal tissues. For the quantitative protein analysis, a Bradford Protein Assay Kit was used. Then, loading a 20 μg protein concentration of each sample with an equal volume of 2x Laemmli sample buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were performed. Separated proteins by SDS-PAGE were then transferred to a membrane called an Immobilon membrane from Millipore. The primary antibodies used were: goat polyclonal antibody p-PI3K p85 alpha (Tyr 508), mouse monoclonal antibody p-AKT1 (Ser 473) and mouse monoclonal antibody p-mTOR (Ser 2448) which were quantitated relative to their total antigens t-PI3K, t-AKT, and t-mTOR, respectively (Santa Cruz Biotechnology, INC. Europe). After blocking the membranes for one-hour, primary antibodies were added to membranes and incubated at 4 °C overnight. At room temperature, appropriate secondary antibodies were incubated for two hours. After washing the membranes twice in 1 x TBS-T, the immunoblots densitometric analysis was done to quantify the quantities of the studied proteins versus the control sample by total protein normalization by using Image analysis software (ChemiDoc MP imaging system, Bio-Rad).

Each homogenized tissue sample was treated with the Ready PrepTM protein extraction kit (total protein) supplied by Bio-Rad Inc. (Catalogue #163-2086) (Bio-Rad Laboratories, Inc. is an American Manufacturer, Hercules, CA, USA) in accordance with the manufacturer’s instructions. Bio Basic Inc. (Markham, ON, Canada) provides the Bradford Protein Assay Kit (SK3041) for quantitative protein analysis. In order to calculate the protein content in each sample, a Bradford assay was carried out in accordance with the manufacturer’s instructions. Then, an equal volume of 2× Laemmli sample buffer containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris HCl was loaded onto each sample’s 20 g protein concentration. When the pH was measured, it was raised to 6.8. To guarantee that the protein was denaturated before loading on the polyacrylamide gel electrophoresis, each combination was cooked at 95 °C for 5 min.