Fig. 1

SDS–PAGE and Western blot analysis of recombinant CD40-N expressed in P. pastoris. a A 20-μL sample of supernatant was loaded onto a 12 % SDS–PAGE gel and stained with Coomassie brilliant blue G-250. All of the SDS-PAGE experiments were performed under the same conditions. Samples were collected at 12-h intervals for 84 h. The sizes of molecular weight markers (kDa) are shown (M), and recombinant CD40-N protein is indicated with an arrow. b Western blot analysis of human Flag-tagged CD40-N (not undergoing codon optimization) and CD40-N-S (with signal sequence and not undergoing codon optimization) expressed in HEK293T cells. pCDNA3.3-CD40-N and pCDNA3.3-CD40-N-S were introduced into HEK293T cells, and the cell lysates were harvested after 48 h. A 10-μl sample was loaded onto a 12 % SDS–PAGE gel. Anti-Flag, anti-CD40-N and anti-GAPDH antibodies were used to detect the proteins. c Western blot analysis of proteins at different times in P. pastoris (0, 12, 24, 36, 48, 60, 72, and 84 h). P indicates the positive control (CD40-N expressed in HEK293T cells)