Gapdh antibody

The IEC-6 cell line and Caco-2 cell line were purchased from the Kunming Cell Bank. IEC-6 cells were at passage 15 and were maintained in T-150 flasks in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated FBS. IEC-6 cells were used at passages 15-20.
High-sugar DMEM cell culture medium was purchased from Hyclone, United States. Fetal bovine serum (FBS), 0.25% Trypsin-EDTA was purchased from BioInd (Biological Industries, Israel). HuR, β-actin antibody was purchased from Cell Signaling Technology, United States. Claudin-1 and Ki-67 antibodies were purchased from Abcam, United Kingdom. GAPDH antibody was purchased from Shenyang Wanlei Biotechnology Co., Ltd. The corresponding secondary antibodies were purchased from Proteintech, United States. PVDF membrane and an immunoblotting chemiluminescence (ECL) system were purchased from Bio-Rad. The reverse transcription kit and Real-time fluorescence quantification kit were purchased from TaKaRa, Japan. siHuR, siSPRY4-IT1, SPRY4-IT1, and Claudin-1 primers were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. The apoptosis detection kit was purchased from BD Biosciences, United States. The cell cycle detection kit was purchased from Beyotime, Beijing. GA (purity ≥ 98%) was obtained from Phystandard Technology LTD (Tianjin, China).

The total protein from mouse corneas (3 corneas/sample/group) and HCE cells was extracted and prepared in a standardized manner for Western blotting. After SDS-PAGE, transfer and blocking with 5% non-fat milk at room temperature for an hour on a shaker, membranes were incubated overnight at 4°C with anti-TRIM21 antibody (Abcam, United Kingdom, used at 1:1000), STING (D2P2F) Rabbit mAb (Cell Signaling Technology, China, 1:1000), IRF-3 Rabbit mAb (Cell Signaling Technology, 1:1000), phospho-IRF3 Rabbit mAb (Cell Signaling Technology, 1:1000) and GAPDH antibody (Wanlei, China, 1:1000) as primary antibodies. Followed by the membranes were washed three times in Tris-buffered saline. Then, the membranes incubated with goat horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Beyotime, China, 1:5000) as secondary reagents for 2 h at room temperature. Finally, an enhanced chemiluminescence kit (Wanlei, China) was utilized to visualize the membrane. The densitometry analysis was performed using ImageJ 6.0 software.