C0001

PROTACs, E3 ligands, MLN8237 (S1133, Selleck), MLN4924 (B1036‐5.1, APExBIO), and Bortezomib (T2399, TargetMol) treated samples as well as released synchronized cells were subjected for immunoblot analysis. Whole cell lysates were extracted using standard Western blot procedures. Fresh cells were lysed on ice in an RIPA buffer (50 × 10⁻³m Tris pH = 8.0, 1 × 10⁻³m EDTA, 150 × 10⁻³m NaCl, 1% NP‐40, 0.5% sodium deoxycholic acid, 0.1% SDS) containing phosphatase and protease inhibitors (C0002 and C0001, TargetMol) for 15 min. Ultrasound was performed at 80 Hz for 5 min and the impurities were removed by centrifuge at 12 000 RPM for 10 min. The concentration of protein supernatant was determined by the Bradford method and an equal amount of protein was subjected for electrophoresis using the procedure of 60V for 30 min followed by 120 V for 1 h. The protein was transferred onto a PVDF membrane with 300 mA for 90 min. After being blocked at room temperature for 1 h with 5% bovine serum albumin (BSA, FC0077, MP), the membrane was incubated overnight with the indicated antibodies at 4 °C. Subsequently, the PVDF membrane was washed three times with TBST and incubated with the peroxidase‐conjugated secondary antibody for 1 h at room temperature. Protein expression was monitored by the BioRad chemiluminescent imaging system with an enhanced chemiluminescence reagent (SQ201, EpiZyme).

The CD4 + T cells co-cultured with hUCMSCs were lysed in RIPA lysis buffer (P0013B, Beyotime, China) containing protease and phosphatase inhibitors (C0001 and C0004, TargetMol, USA). A total of 10 µg protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 3% BSA in PBS for 2 h, the membrane was incubated with primary antibodies against phospho-signal transducer and activator of transcription (STAT) 6 (1:1000, Abcam, ab263947), STAT6 (1:1000, Proteintech, 10253-2-AP), or GAPDH (1:5000, Proteintech, 60004-1-Ig) for 12 h, then incubated with anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5000, Proteintech, SA00001-2) for 1 h at room temperature. The blots were visualized with a chemiluminescence imaging system (Tanon 5200, Shanghai, China) and quantified using ImageJ (Version 1.48v).

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China) containing protease inhibitors and phosphatase inhibitors (C0001 and C0004, TargetMol, Massachusetts, USA). Total lysates were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE, C671102, Sangon Biotech) and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00005, Millipore, Burlington, MA, USA). The membranes were incubated with GMFB-specific antibodies (1:200, SP-61, Santa Cruz) and visualized with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000). The corresponding bands were detected using an enhanced chemiluminescent (ECL) detection kit (36208ES60, Yeasen).