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. 2014 Aug 19;9(8):e105409. doi: 10.1371/journal.pone.0105409

Figure 5. Native human plasma and RPE CFH is sulfated.

Figure 5

(A) About 4 µg of purified human plasma CFH was either untreated (lane 1) or treated with PNGase F (lane 2), and then immunoblotted with anti-CFH antibody or with PSG2. CFH was also immunoprecipitated from human RPE, and was either untreated (lane 1) or treated with PNGase F (lane 2), and then immunoblotted with anti-CFH antibody or with PSG2. Western blots were repeated 3 independent times from human plasma and RPE samples. (B) A recombinant human CFH clone was transfected into HEK293T cells, immunoprecipitated with anti-CFH antibody, and was either directly electrophoresed (lane 1) or first treated with PNGase F (lane 2). The immunoprecipitants were immunoblotted with anti-CFH antibody or with PSG2. Immunoprecipitation and Western blots were repeated 3 independent times after CFH transfections. (C). Recombinant CFH was transfected into HEK293T cells and radiolabelled with radioactive sulfate, then immunoprecipitated and subjected to PNGase treatment (lane 3) or left untreated (lane 2). The blots were autoradiographed (AR), then immunoblotted with anti-CFH antibody. (D). Radiolabelled CFH was analyzed by barium hydroxide hydrolysis and autoradiographed (AR). TLE experiments were repeated at least three independent times.