Ecl plus

Proteins of total cell lysate or proteins from the tissue were extracted using RIPA lysis and extraction buffer. After brief sonication and centrifugation, protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). A total of 10 μg protein was electrophoresed on a 10% sodium dedecyl sulfate polyacryamide gel and subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 hour. The membrane was then incubated with the desired primary antibodies (1:1,000 dilution) at 4°C overnight. The next day, the membrane was washed 3 times with TBST, followed by incubation with horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody (1:4,000, Rockland, Limerick, PA) at room temperature for 1 hour. After washing 3 times with TBST, the membrane was developed using ECL Plus (EMD Millipore, Billerica, MA), and exposed to X-ray film. β-Actin was used as an internal loading control.

A RIPA kit (Beyotime Institute of Biotechnology) was used to obtain the whole protein from cells and tissues. The protein concentration was measured via BCA Protein Assay kit (Beyotime Institute of Biotechnology). Then, 10% SDS-PAGE was used for electrophoresis with 20 µg protein in each lane. Protein was transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked in 10% BSA solution (Beyotime Institute of Biotechnology) at room temperature for 2 h and incubated in an incubation box at 4°C with the primary antibodies overnight. The membranes then were washed with TBS with Tween-20 (5% TBST) two or three times every 10–15 min. Subsequently, the membranes were incubated with immunoglobulin G (anti-mouse or rabbit) at room temperature for 2 h and washed with TBS + 0.001% Tween-20 buffer two or three times again, each time lasted 10–15 min. ECL Plus (EMD Millipore) was adopted for detection of protein expression levels by Bio-Imaging System. The primary antibodies included were: DNAJC2 (1:2,000; cat. no. ab231318), P21 (1:5,000; cat. no. ab109520; both Abcam), AKT (1:2,000; cat. no. 10176-2-AP) and p-AKT (1:3,000; cat. no. 66444-1-Ig; both from ProteinTech Group, Inc.). Secondary antibodies were goat anti-rabbit IgG (H+L; 1:5,000; cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc.).

Total protein extraction was performed using the RIPA kit (Beyotime, Shanghai, China). The extracted proteins were then subjected to SDS-PAGE for separation, followed by transfer onto PVDF membranes. Subsequently, the membranes were incubated overnight at 4 °C with specific antibodies. β-actin was utilized as a loading control. ECL Plus (EMD Millipore, Billarica, MA, USA) was employed to visualize the ECL signals.