Sds page sample loading buffer 6x

Total protein from the jejunum was extracted using RIPA cell lysis buffer (Beyotime, Beijing, China). Nuclear proteins were extracted using a Minute Cytosolic and Nuclear Extraction Kit for Frozen/Fresh Tissues (Invent Biotechnologies, Beijing, China). The protein concentration was estimated using BCA Protein Assay Kit (Beyotime). Samples were mixed with SDS-PAGE Sample Loading Buffer 6X (Beyotime), and incubated for 10 min at 100°C.

After transfection for 48 h, cells were washed with PBS and lysed with RIPA Lysis Buffer (P0013B, Beyotime Biotechnology) containing protease inhibitors. SDS‐PAGE Sample Loading Buffer 6X (Beyotime Biotechnology) was added to the cell samples, and proteins were denatured by boiling at high temperature. After the samples were cooled, SDS‐PAGE protein gel electrophoresis was performed. A 6% SDS‐PAGE gel (P0012A, Beyotime Biotechnology) was prepared, and the extracted protein samples were separated by gel electrophoresis, transferred to PVDF membranes, blocked with 10% skimmed milk powder for 2 h at room temperature, washed with TBST, and then incubated overnight at 4°C with GFP primary antibody (50430, Proteintech) diluted 1:2000. The membrane was washed with TBST, and horseradish peroxidase (HRP)‐labeled goat anti‐rabbit secondary antibody (A0208, Beyotime Biotechnology) diluted 1:1000 was added and incubated for 2 h at room temperature. Finally, the membrane was exposed to ultrasensitive ECL reagent (P0018, Beyotime Biotechnology) for color development, and the protein bands were imaged by a high‐sensitivity chemiluminescence ChemiDoc XRS system (Bio‐Rad).