Hrp conjugated affinipure goat anti mouse rabbit igg

The following reagents and antibodies were used in this study: DMEM (GIBCO BRL); fetal bovine serum (FBS, GIBCO BRL, 10099141); Lipofectamine® 3000 reagent (L3000015, Invitrogen, Thermo Fisher Scientific, USA); TRIzol reagent (TaKaRa, 15596018, China); PrimeScript™ RT reagent Kit (TaKaRa, RR037Q, China); CCK-8 kit (Meilune, MA0218, China); DMEM-diluted Matrigel (BD Biosciences, USA); 5% propidium iodide (Med chemExpres, China); Annexin V-PE and PI (Med chemExpres, China); 10% SDS-PAGE (Beyotime, China); PVDF membrane (Bio-Rad Laboratories, USA); 5% skimmed milk (BBI, China); HRP-conjugated Affinipure Goat Anti-Mouse/Rabbit IgG (Proteintech, SA00001-1/SA00001-2, China). The primary antibodies for western blotting were provided in Supplementary Table 1.

We lysed the HepG2 cells transfected with si-SNRPD1 or si-NC using RIPA buffer (Meilunbio, China) and incubated on ice for 30mins then extracted the total protein. We separated the total protein by 10% SDS-PAGE (Beyotime, China), then transferred the separated protein to PVDF membrane (Millipore, USA), and then blocked it in 5% non-fat skimmed milk powder (BBI, China) at room temperature for 2 hours. Next, we incubated the membranes with the primary antibodies (SNRPD1: Abcam, ab233115, UK; mTOR: Proteintech, 66888-1-Ig, China; LC3: Abcam, ab192890, UK) overnight at 4°C. Thereafter, we incubated the membranes with the second antibodies (HRP-conjugated Affinipure Goat Anti-Mouse/Rabbit IgG: Proteintech, SA00001-1/SA00001-2, China) for 1 h at 25 °C. Finally, we used the chemiluminescence imaging system (BIO-RAD, USA) to detect the protein blots, and Image Lab software (BIO-RAD, USA) was used to measure the protein expression value.