Figure 4

MIF/A1-I3 complexes in bladder homogenates. MIF WB under native, non-reducing and reducing conditions (A) showed that a broad band approximately 60–500 kDa under native conditions resolved into 4 distinct bands under non-reducing conditions. These bands had the apparent molecular weight of 170, 130, 75 and 12 kDa. The 12-kDa band corresponds well with monomeric MIF. Under reducing conditions, only two high-molecular weight MIF bands with apparent molecular weights of 130 and 75 kDa are present, in addition to the 12 kDa band. (B) A1-I3 WB under non-reducing conditions showed correspondence to the high-molecular weight MIF bands (170, 130 and 75) indicating MIF/A1-I3 complexes. In addition, a 200-kDa A1-I3 band (marked by asterisk) was not detected under MIF WB, indicating that this form of A1-I3 is not bound to MIF. Substance P treatment appeared to decrease the 170-kDa MIF band, while increasing the intensity of the 200-kDa and 170-kDa A1-I3 bands. Under reducing conditions, an A1-I3 band at 100 kDa (marked by asterisk) can also be observed. For non-reducing and reducing WB, representative WB from two different animals in each group are presented. Apparent molecular weights (in kDa) are indicated by numbers to the right of the lanes.