A4782

Tianjin Institute of Urology (Tianjin, China) provided the Caki-1, A498 and OSRC-2 cell lines. ENCODE cell culture standards were followed when growing the cells.
The primary anti-SAA1 (A1655, ABclonal, China), anti-ERK1/2 (A4782, ABclonal, China), anti- Phospho-ERK1-T202/Y204 + ERK2-T185/Y187 (AP0974, ABclonal, China), anti-c-Jun (A0246, ABclonal, China), and anti-ß-Actin (AC026, ABclonal, China) antibodies were used according to the instructions.

Liver and intestinal tissues were lysed in RIPA buffer containing protease inhibitors (Solarbio Science & Technology Co., Ltd., Beijing, China) and phosphatase inhibitors (Applygen Technologies Inc., Beijing, China). After centrifugation, the protein concentration of the lysate was determined with a BCA protein assay kit (P0012, Beyotime, Shanghai, China). An appropriate amount of protein was separated on SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked after 1.5 h with 5% skim milk and incubated with antibodies against ZO-1 (Ab96587, Abcam, Cambridge, UK), Claudin-1 (Ab180158, Abcam), Occludin (Ab216327, Abcam), MEK1/2 (A4868, ABclonal, Wuhan, China), p-MEK1/2 (AF3385, Affinity Biosciences, Jiangsu, China), ERK1/2 (A4782, ABclonal), p-ERK1/2 (AP0234, ABclonal), pIgR (A6130, ABclonal), or GAPDH (A6130, Proteintech, Wuhan, China) overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated second antibodies against mouse or rabbit IgG (LF102 and LF101, respectively, Epizyme, Inc., Shanghai, China) for 1 h at 25 °C. Protein bands were visualized using a Bio-Rad immunoblot analysis detection system (Bio-Rad Laboratories, Hercules, CA, USA). Western blot band quantification was performed using ImageJ (Bethesda, MD, USA) software and normalized to GAPDH.

Western blotting assays were conducted as previously reported [18 (link)]. The primary antibodies were as follows PR55α (1/1000, A2185, Abclonal, China), AKT (1/1000, A17909, Abclonal, China), p-AKT-T308 (1/1000, AP0304, Abclonal, China), P-AKT-S473 (1/1000, AP0140, Abclonal, China), ERK1/2 (1/1000, A4782, Abclonal, China) and p-ERK1/2-T202/Y204 (1/1000, AP0472, Abclonal, China).

The cells were lysed with RIPA buffer (Beyotime, China) containing protease inhibitors (MCE, USA) and phenylmethanesulfonyl fluoride (PMSF) (Sigma, USA). Protein concentrations were determined using a BCA protein assay kit (Beyotime, China). Proteins were separated using 10% or 12% SDS-PAGE, transferred to PVDF membranes (Roche, Switzerland), and blocked with 5% skim milk (BD, USA) for 1 h. After washing, the samples were incubated with the primary antibody overnight at 4 °C. The next day, the PVDF membrane was incubated with the secondary antibody for 2 h at room temperature, and the protein level was detected using the ChemiDoc XRS + imaging system (Biorad, USA). The primary antibody was used at a 1:1000 dilution. Antibody product numbers are as follows: anti-OGDHL (A15475, ABclonal, China), anti-FASN (A19050, ABclonal, China), anti-TFAP2A (13019-3-AP, Proteintech, China), anti-FTO (A3861, ABclonal, China), anti-RUNX1 (A0400, ABclonal, China), anti-ARNT (A0972, ABclonal, China), anti-CyclinD1(A2708, ABclonal, China), anti-MMP2 (A19080, ABclonal, China), anti-MMP9 (A0289, ABclonal, China), anti-pERK (AP0485, ABclonal, China), anti-ERK (A4782, ABclonal, China), anti-GAPDH (A19056, ABclonal, China). The original blots of the western blotting can be found in the Original data files.