Pcmv myc

Human embryonic kidney (HEK) 293 cells were maintained in DMEM (Invitrogen). The media was supplemented with 10% FBS, 100 U/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Polyclonal antibodies against the epitope tags (Flag, HA, and Myc), Ubiquitin, BLIMP1, and β-actin were obtained from Santa Cruz Biotechnology, Inc. Anti-Hrd1 and anti-Tubulin were purchased from Sigma-Aldrich. Fluorescence-labeled antibodies, including CD11c, CD11b, CD4, CD8, CD45.1, CD45.2, MHC-I, MHC-II, CD80, and CD86, were used for flow cytometry analysis (eBioscience). Hrd1 and the Ubiquitin expression plasmids were obtained as reported previously (Gao et al., 2008 (link)). Flag-BLIPM1 expression plasmids were purchased from Addgene. The truncation mutants of both Hrd1 and BLIMP1 were generated by PCR and subcloned into pCMV-Flag (Sigma-Aldrich) or pCMV-Myc vectors (Invitrogen).

Human cells were cultured in standard cell culture Dulbecco's modified Eagle's medium at 37 °C in a humidified incubator with 5% CO₂(v/v). The human METTL3 gene was cloned into pCMV-Myc (Invitrogen), S-protein/FLAG/SBP (streptavidin-binding protein) triple-tagged destination vector^{48 (link)} or pGEX-5×-2 (GE healthcare). The human WTAP gene was cloned into p3XFLAG-CMV-14 (Sigma) or pProEX-HTb (Invitrogen). The human METTL14 gene was cloned into pcDNA3-HA (Invitrogen). The following antibodies were used: rabbit-anti-METTL3 (Abcam), mouse-anti-METTL3 (Abnova), mouse-anti-WTAP (Santa Cruz), rabbit-anti-METTL14 (Atlas), rabbit-anti-m6A (Synaptic Systems) and other antibodies included in Supplementary information, Data S1.

Total RNA extracted from HEK293T cells was used for cDNA synthesis by SuperScript® III Reverse Transcriptase (Invitrogen) with a random primer. The obtained cDNA was used as a template to amplify the U1 snRNP proteins and the 3′ end processing factors with Phanta Max Super-Fidelity DNA Polymerase (Vazyme). Target fragments were cloned into pCMV-MYC, pCMV-FLAG, pmCherry-C1, and pEGFP-C1 expression vectors (Invitrogen) with ClonExpress® Ultra One Step Cloning Kit (Vazyme). The mutant vector MYC-SNRNP70-Mut was constructed by Tsingke. All constructed expression plasmids were confirmed by Sanger sequencing (Tsingke).