Fig 1: Western blots of secreted (a) and intracellular (b) protein fractions of Expi293F cells expressing WT and B.1.351 RBDmfc together with increasing amounts of PDIA2. Secreted and intracellular protein fractions described in Figure 3 were analysed by Western blot using antibodies directed against the recombinant RBDmfc proteins (anti-RBD, anti-Fc), against chaperones (anti-PDIA2, anti-HSPA5, anti-HSP90B1), and against ßactin as loading control. M, protein ladder (apparent MW in kDa indicated on the left); *, endogenous PDIA2 protein (anti-PDIA2 blot); arrow, RBDmfc proteins (anti-RBD and anti-Fc blots in panel (b)) likely lacking disulphide bonds and mature glycosylations, and thus running in SDS-PAGE with an apparent MW of 50 kDa (compare to 59 kDa reduced, mature proteins shown in Figure 2, lanes 3 and 4). Quantification of signal intensity of chaperones is shown in Figure S2. Data are from one representative experiment (out of three independent experiments). Uncropped blot images are shown in Appendix A Figure A3 and Figure A4.
Fig 2: Concentration of intracellular and secreted recombinant RBDmfc proteins upon co-transfection of Expi293F cells with PDIA2-expressing or empty vectors. Expi293F cells were transfected with 1 µg plasmid DNA per ml cell suspension (“100%” thus corresponding to 1 µg plasmid DNA). In co-transfection experiments, the respective proportion of plasmid expressing RBDmfc, PDIA2 or empty plasmid (to complete to 1 µg plasmid DNA) is indicated below each bar. Accordingly, 100%, 95%, 70% or 5% plasmid DNA indicates that 1, 0.95, 0.7 or 0.05 µg plasmid DNA (per ml cell suspension) was used for transfection, respectively. Intracellular and secreted protein fractions were prepared four days after transfection. Protein concentration was measured by Octet RED96 and expressed in µg/mL culture. Values are mean (standard deviation) of duplicate Octet RED96 measurements of one representative experiment (out of two independent experiments).
Supplier Page from Sino Biological, Inc. for Human PDIA2 Gene ORF cDNA clone expression plasmid