Figure 4

Western blot and ELISA analysis of HMBS and ATP2C1. (a) Cell lysates were applied to SDS-PAGE gels under reducing conditions. HMBS and ATP2C1 protein were detected in platelets using a polyclonal antibody. Incubation of platelets with E. coli K12 and E. coli O18:K1 in 1:5 or 1:10 platelet-bacteria ratios converted HMBS 47 kDa form to a 40 kDa protein. ATP2C1 was not affected. GAPDH was used as a loading control. (b) The releasates of the platelet-bacteria mix were collected after centrifugation (500 g, 10 minutes without break). HMBS, ATP2C1 and LRCH4 levels were measured by ELISA. Data represents the mean of three independent experiments (n = 3). ATP2C1 was detectable in platelet supernatants, while HMBS and LRCH4 proteins were either not released from platelets or in concentrations below the detection level of the ELISA (data not shown). BDL, below detection limit. Western blot results are representative image of three replications. The same exposure was applied equally across the entire image. The original pictures of the full-length western blots can be found in Supplementary Fig. S7a, b, c, d, g, h.