Sds page buffer

Cells were washed with cold PBS and lysed in RIPA buffer (#R0278, Sigma-Aldrich) containing protease and phosphatase inhibitor cocktails (Roche). The total protein concentration in the lysates was determined using the BCA kit (#BCA1, Sigma-Aldrich). The proteins were mixed with 5X SDS-PAGE buffer (Biosesang), separated using SDS-PAGE (8% or 12%), and then transferred to nitrocellulose or PVDF membranes (Bio-Rad, Hercules, CA, USA) at 70 V for 60 min. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing Tween-20 (TBST) for 1 h at 4 °C to prevent nonspecific binding. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies diluted at 1:500 or 1:1000. After washing with TBST, the membranes were incubated with the appropriate secondary antibodies (Sigma-Aldrich) for 1 h at 25 °C. The immunoblots were developed using a chemiluminescent substrate (Thermo Fisher Scientific, Sunnyvale, CA, USA). Protein expression was then quantified using ImageJ (version 1.54d, NIH, Bethesda, MD, USA).

Cells were washed with DPBS and lysed with lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland). The lysate was centrifuged, and proteins were separated from lysate mixed with 5X SDS-PAGE buffer (Biosesang, Yongin, Republic of Korea) using SDS-PAGE and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). After treatment with a blocking buffer (Bio-Rad, Hercules, CA, USA) to prevent nonspecific binding, the membranes were incubated with primary antibodies overnight at 4 °C, followed by washing with TBST and further incubation with secondary antibodies for 1 h at 25 °C. The proteins were detected using chemiluminescent substrate (Thermo Fisher Scientific, Sunnyvale, CA, USA), and densitometry was conducted using ImageJ (version 1.54d).