Fig. 1: Fortilin and TGF-βs specifically interact with each other.
From: Fortilin interacts with TGF-β1 and prevents TGF-β receptor activation
IP, immunoprecipitation; IB, immunoblot; α-Flag, anti-FLAG antibody; α-TGF-β1, anti-TGF-β1 antibody; α-His6, anti-hexa-histidine antibody; ELISA, enzyme-linked immunosorbent assay; TMB, 3,3’,5,5’-tetramethylbenzidine; HRP, horse-radish peroxidase; Capture, capturing antibody; Detection, detecting antibody; SeraTGF-β1+, fortilin+, sera generated from platelet-rich plasma; SeraTGF-β1-, fortilin-, SeraTGF-β1+, fortilin+ immunodepleted of both fortilin and TGF-β1; BSA, bovine serum albumin; Ab, antibody; ABS450, absorbance at 450 nm; Data points, means ± SD; statistical analyses performed using ANOVA with Fisher’s multiple comparison; ****P < 0.001. a Fortilin interacts with TGF-β1. Recombinant Flag-tagged fortilin, TGF-β1, Flag-tagged p53, and His6-tagged NQO2 were incubated in binding buffer. α-fortilin antibody was used to immunoprecipitate fortilin, and immune complexes were resolved in SDS-PAGE and blotted onto a nitrocellulose membrane. Immunoblot analyses using α-TGF-β1, α-Flag, and α-His6 antibodies showed that fortilin co-immunoprecipitated TGF-β1 and p53 but not NQO2. Fortilin is known to interact with p53 but not with NQO2. INPUT represented 10% of the total reaction mixture used for IP. b Fortilin interacts with TGF-β2 and -β3. Recombinant Flag-tagged fortilin, TGF-β2 or TGF-β3, and His6-tagged NQO2 were incubated in binding buffer. α-fortilin antibody was used to immunoprecipitate fortilin, and immune complexes were resolved in SDS-PAGE and blotted onto a nitrocellulose membrane. Immunoblot analyses using α-Flag, α-TGF-β, and α-His6 antibodies showed that fortilin co-immunoprecipitated TGF-β2 and -β3, but not NQO2. INPUT represented 10% of the total reaction mixture used for IP. c Detection of TGF-β1 in normal human serum. The TGF-β1 ELISA that used α-TGF-β1 capturing and detecting Abs showed high TGF-β1 levels in SeraTGF-β1+, fortilin+ (column 1) compared with those in SeraTGF-β1-, fortilin- (column 2). N = 3. d Detection of fortilin in normal human serum. The in-house fortilin ELISA that used α-fortilin capturing and detecting Abs showed high fortilin levels in SeraTGF-β1+, fortilin+ (column 1) compared with those in SeraTGF-β1-, fortilin- (column 2). N = 3. e Detection of the fortilin-TGF-β1 interaction in normal human serum. The modified ELISA system that used α-TGF-β1 capturing and α-fortilin detecting Abs yielded a robust signal in SeraTGF-β1+, fortilin+ (column 1) compared with those in SeraTGF-β1-, fortilin- (column 2). N = 3.
