FIGURE 4

Expression of endogenous and recombinant ParvB protein. (a) To determine specificity, affinity purified ParvB antibody (raised against recombinant ParvB3 residues 1–142, including the common CH1 domain) was incubated with recombinant ParvB3 or GST protein, then used to probe 50 g HEK293 cell lysates resolved on 12% SDS–PAGE. The anti-ParvB antibody specifically detected two endogenous protein bands (45 and 40 kDa) that can be absorbed by ParvB3-GST fusion peptide. (b) Cellular proteins (50 g/lane) from parental and EGFP-ParvB3 HEK293 transient transfectants were resolved by 12% SDS–PAGE. Affinity purified ParvB antibody detected the exogenous ParvB3 and CH1 EGFP proteins, in addition to the endogenous ParvB protein bands. The membranes were subsequently probed with GFP and GAPDH antibodies. (c) Domain structure of ParvB3. Full-length ParvB3 cDNA (residues 1–316) and two truncated variants, CH 1 and CH 2, corresponding to residues 1–142 and 157–316, respectively, were cloned into pEGFP-C3 vector as described in Materials and methods