Nonfat dry milk

HEK-293T cells were co-transfected with 1 μg of pCMV-myc expressional vector encoding Vif variants including three Vif C variants (VifS1, VifS3 and VifS17), two Vif B variants (VT3 and VT4) and four Vif B/C recombinants (D43, E43, D48 and E48), and 1 μg of pCMV-HA-A3G. pCMV-HA-A3G alone was used as control. After 36 hours of co-transfection, cells were harvested and lysed in RIPA lysis buffer (1% NP-40, 20 mM TrisCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Sodium deoxycholate, 1 mM Na3VO4). Protein concentration was measured using BCA protein assay kit (Pierce, Thermo Scientific). Equal amount of proteins were resolved by 10% SDS PAGE (Sodium Dodecyl Sulfate- Poly-Acrylamide Gel Electrophoresis) and were transferred to nitrocellulose membrane. The membranes were blocked with 1% BSA (Bovine Serum Albumin; sigma) and 5% non fat dry milk (Himedia Laboratories) in 1X PBS (Phosphate Buffer Saline) and washed thrice with 1X PBS containing 1% Tween 20 (PBST; MERCK). Then the membranes were probed with primary antibody (Anti-Myc antibody, Anti-Vif antibody and Anti-HA antibody) washed with PBST and probed with horseradish peroxide (HRP)-conjugated secondary antibody (Anti-mouse antibody). Blots were developed using ECL (Enhanced Chemi-luminiscent) reagent. GAPDH was used as a loading control in all cases (as described47 48 (link)49 (link)).

Rats were decapitated with an overdose of pentobarbital at 24 h after reperfusion. Ipsilateral brains were excised and homogenized in RIPA buffer (150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris–HCl (pH-7.4), 10% glycerol, 1% NP40, and 0.5% sodium deoxycholate). Protein concentration in the supernatant was determined by the Bradford method following centrifuging tissue homogenates at 10,000 rpm for 20 min. An equal amount of total protein sample (75 μg) was probed by western blots. Total protein extract was resolved on 8% and 10% SDS-PAGE gels and transferred on to nitrocellulose membrane. The membranes were incubated with 5% nonfat dry milk (HIMEDIA, India) in tris-buffered saline containing Tween-20 (TBS-T) for 90 min at room temperature (RT). The membranes were incubated overnight at 4 °C with primary antibody (1:1000 dilution) against SIRT1, phospho and total-JNK, phospho and total-ERK, phospho and total-MEK, phospho and total-AKT, beta-tubulin (Cell Signaling Technology, USA), and phospho-IRS-1 (ser307) (Merck, USA). Membranes were washed with TBS-T and incubated with HRP labeled secondary antibody (CST, USA) specific for mouse and rabbit for 90 min at 37 °C (1:3000 dilution). The chemiluminescence was captured using photographic film and ECL detection system (Bio-Rad, USA).

HEK-293T cells were transfected with the gene of interest for either 24 h or 36 h. The cells were harvested and lysed in radioimmunoprecipitation assay lysis buffer (1% NP-40, 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% sodium deoxycholate, and 1 mM Na₃VO₄). Protein estimation was carried out by using BCA Protein Assay Kit (Pierce, Thermo Scientific). An equal amount of protein was loaded on SDS-PAGE and transferred to nitrocellulose membrane as described before (24 (link)). The membranes were blocked with 5% nonfat dry milk (Himedia Laboratories) or 5% bovine serum albumin. The primary antibodies used were anti-AKT, anti-GAPDH, anti-phospho-AKT (S-473) (Cell Signaling Technology), anti-Myc, anti-HA (Clontech), ab140601 (abcam–K48 antibody), and anti-GST (Santa Cruz Biotechnology). The secondary antibodies used were anti-rabbit/mouse-horseradish peroxidase conjugated (Jackson ImmunoResearch). Blots were developed using Enhanced Chemiluminescence) reagent.