Hrp conjugated secondary antibodies

Total RNA was extracted using RNAiso Plus (Takara, China) according to the manufacturer’s protocol. Quantitative PCR analysis of PRMT5-ISO5 was carried out with 10 μL TB Green™ Premix Ex Taq™ II (Takara, China) by the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The primers for RT-qPCR are shown in Supplementary Table S1.
Proteins extracted from cells and tissues were measured and heat denatured. Equal amounts of proteins were separated using 10% or 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Boston, MA, USA). After saturating, the membranes were incubated with anti-PRMT5 (ab109451, Abcam, Boston, MA, USA), anti-HNRNPH1 (ab10374, Abcam, Boston, MA, USA), anti-SRSF3 (ab198291, Abcam, Boston, MA, USA), anti-AKT1 (75692S, CST, Boston, MA, USA), anti-phospho-AKT (Ser473) (4060S, CST, Boston, MA, USA), anti-SDMA (13222S, CST, Boston, MA, USA) or anti-GAPDH (60004-1-Ig, Proteintech, Shanghai, China), and subsequently incubated with HRP-conjugated secondary antibodies (Biosharp, Guangzhou, China). Finally, the membranes were detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and visualized with ChemiScope 6100 (CliNX Science Instruments, China).

Cell pellets or intestinal tissues were lysed with RIPA buffer (Beyotime, China) for 20 min on ice and lysates were collected by centrifugation (13000 × g, 20 min at 4°C). Protein concentrations were quantified using the BCA Protein Assay (Thermo Fisher Scientific, USA) and equal amounts (20 μg) of protein lysates from each sample were separated by SDS-PAGE. Separated proteins were then transferred to PVDF membranes (Millipore, USA). Membranes were blocked with 5% skim milk or BSA and then incubated with the following primary antibodies: anti-TLR4 (Abcam, USA), anti-p-IκBα (Abcam, USA), anti-IκBα (Abcam, USA), anti-p-PI3K (Cell Signaling Technology, USA), anti-p-AKT (Cell Signaling Technology, USA), anti-PI3K (Cell Signaling Technology, USA), anti-AKT (Cell Signaling Technology, USA), anti-β-actin (Abcam, USA). Membranes were then incubated with species-matched HRP-conjugated secondary antibodies (Biosharp, China). Protein expressions were visualized using Immobilon^® Western Chemiluminescent HRP Substrate (Millipore, USA) in accordance with the manufacturer’s protocol.

Cells were lysed on ice with 200 μL Radio Immunoprecipitation Assay (RIPA; Beyotime, Shanghai, China) lysis buffer containing 1% proteinase inhibitor (PMSF; Beyotime, Shanghai, China). The cell lysate was centrifuged at 15,000 rpm for 15 min at 4 °C and the concentration of protein samples was determined using the BCA assay kit (Beyotime, Shanghai, China). Then, samples containing 50 μg protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE; GenScript, Nanjing, China) and transferred to the polyvinylidene difluoride membrane (PVDF; Life Technologies Carlsbad, CA, USA). Membranes were blocked with 5% free-fat milk in Tris-buffered saline with tween (TBST; Solarbio, Beijing, China) for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C (Table 2). After three washes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (Biosharp, Shanghai, China) for 2 h at room temperature. Finally, the signals were detected by the enhanced chemiluminescence substrate kit (ECL; Biosharp, Shanghai, China) and exposed to the chemiluminescence detection system (Amersham, Piscataway, Nanjing, China) following company guidelines. Immunoblots were scanned and densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).