Nupage sds gel system

Cells were lysed in lysis buffer (1% NP‐40, 150 mM NaCl, 20 mM Tris‐HCl, pH 7.5, 0.5 mM EGTA, and 0.1 mM DTT) containing cOmplete mini protease inhibitor cocktail tablets (Merck) and phosphatase inhibitor cocktail (Nacalai Tesque). Cell lysates were boiled for 10 minutes with 4× NuPAGE LDS sample buffer (Thermo Fisher Scientific). Proteins were separated using the NuPAGE SDS gel system (Thermo Fisher Scientific), electroblotted onto a PVDF membrane (Amersham Hybond‐P; Cytiva) and subjected to immunodetection using the following primary Abs at 1:1000 dilution; monoclonal mouse antibodies: anti‐α‐tubulin B‐5‐1‐2 (T5168; Merck), anti‐Cyclin B (610219; BD Biosciences), and anti‐Bcl‐2 (sc‐509; Santa Cruz Biotechnology); polyclonal rabbit antibodies: anti‐CHAMP1 (HPA008900; Atlas Antibodies), anti‐Mcl‐1 (4572; Cell Signaling Technology), anti‐Bcl‐xL (2762; Cell Signaling Technology), anti‐MAD2L2 (12683‐1‐AP; Proteintech), and anti‐Bak (3814; Cell Signaling Technology); and monoclonal rabbit antibody: anti‐Bim (2933; Cell Signaling Technology). Blocking and Ab incubations were carried out in 3% nonfat dry milk. Proteins were visualized using HRP‐labelled secondary Abs (1:5000; Santa Cruz Biotechnology) and enhanced chemiluminescence using ECL Prime Western Blotting Detection Reagents (Cytiva), according to the manufacturer’s instructions.

Cells were lysed in TNE-N buffer (1% NP-40, 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA). Protein concentration in the cell lysate was measured using the Bio-Rad Protein assay kit. Cell lysates were boiled for 10 min with 4×NuPAGE LDS sample buffer (Thermo Fisher Scientific). Proteins were separated using the NuPAGE SDS gel system (Thermo Fisher Scientific), electroblotted onto a polyvinylidene difluoride membrane (Amersham Hybond-P; GE Healthcare Life Sciences), and subjected to immunodetection using appropriate primary antibodies. Blocking and antibody incubations were performed in 3% nonfat dry milk. Proteins were visualized using horseradish peroxidase–labeled secondary antibodies (Santa Cruz Biotechnology; 1:3,000) and enhanced chemiluminescence using ECL prime Western Blotting Detection Reagents (GE Healthcare Life Sciences) according to the manufacturer’s instructions.

Proteins were extracted using a post-alkaline extraction method in accordance with the method of Kushnirov (Kushnirov, 2000 (link)). Briefly, exponentially growing cells (10 mL culture, OD₆₀₀ = 0.3–0.8) were treated with 200 μL of 0.1 M NaOH for 5 min, and then the cell pellet was collected by centrifugation. The cell pellet was treated with sample buffer (60 mM Tris-HCl (pH 6.8), 5% glycerol, 2% SDS, 4% 2-mercaptoethanol and 0.0025% bromophenol blue) at 95°C for 5 min. Crude extracts were cleared by centrifugation, and the supernatant was subjected to western blotting with specific antibodies for analysis. Chemiluminescence signals for horseradish peroxidase (HRP) were detected using an image analyzer (LAS3000mini or LAS4000mini, Fuji, Tokyo, Japan). All western blotting experiments were done independently at least twice to confirm the reproducibility of the results.
To check the efficiency of RNAi in human cells, cells were lysed in TNE-N buffer (1% NP-40, 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA), and were boiled for 10 min with 4×NuPAGE LDS sample buffer (Thermo Fisher Scientific). Proteins in cell lysate were separated using the NuPAGE SDS gel system (Thermo Fisher Scientific) and subjected to western blotting with specific antibodies for analysis.