Culture maintenance, plasmid transfection and BCR stimulation of chicken DT40 B cell lines were carried out as previously described46 (link). Jurkat cell culture and transfection techniques were also performed as described8 (link). The SOS1−2−deficient DT40 B cells were generated in Dr. Tomohiro Kurosaki’s laboratory (RIKEN). Both wildtype and SOS1−2− deficient DT40 B cells were gifts from Dr. Kurosaki. Obtained cell lines were confirmed to be free of mycoplasma contamination. For routine cell functional authentication, surface expression of B cell receptor (BCR) was confirmed by flow cytometry and by BCR-induced pERK2 measurement similar to the experiment shown in Supplementary Figure 4 . Jurkat T cells were obtained from ACCC and were maintained according to the provided guideline.
To generate EGFP-tagged hSOS1 variants, EGFP coding sequence (CDS) was PCR-amplified with Xba I- and Not I-flanked primers from pEGFP-N1 plasmid (Clonetech). Resulting SOS1-EGFP construct bears a 5 amino acid linker (SRGGR) between SOS1 and EGFP CDS. Expression was confirmed by Western blotting with anti-GFP antibody (Supplementary Fig. 4a ).
To generate EGFP-tagged hSOS1 variants, EGFP coding sequence (CDS) was PCR-amplified with Xba I- and Not I-flanked primers from pEGFP-N1 plasmid (Clonetech). Resulting SOS1-EGFP construct bears a 5 amino acid linker (SRGGR) between SOS1 and EGFP CDS. Expression was confirmed by Western blotting with anti-GFP antibody (
