Figure 1.
Figure 1. FIEL1–PIAS4 pathway in pulmonary fibrosis. (A) PIAS4 protein half-life determination in MLE cells transfected with empty plasmid or ubiquitin plasmid (n = 2). (B) PIAS4 protein half-life determination with MG132 or leupeptin treatment (n = 3). (C) Immunoblots (top) showing levels of PIAS4 protein and V5 after KIAA0317 (823 aa, AREL1) and (789 aa, FIEL1) plasmid expression. PIAS4 protein quantification was normalized and graphed (bottom). Data represent mean values ± SEM (n = 3 independent experiments; *, P < 0.05 compared with 0 µg plasmid, Student’s t test). (D) PIAS4 protein was immunoprecipitated from cell lysate using a PIAS4 antibody and coupled to protein A/G beads. PIAS4 beads were then incubated with in vitro–synthesized products expressing HIS-V5-FIEL1 (789 aa) or HIS-V5-AREL1 (823 aa). After washing, proteins were eluted and processed for V5 immunoblotting (n = 2). (E) In vitro ubiquitination assay. Purified E1 and E2 components were incubated with V5-PIAS4 and FIEL1. The full complement of ubiquitination reaction components (second lane) showed polyubiquitinated PIAS4 proteins (n = 3). (F) Immunoblots showing levels of PIAS proteins and V5 after ectopic FIEL1 or UBE3B expression. (G and H) PIAS4protein half-life determination in MLE cells with empty plasmid or FIEL1 expression (G); PIAS4 protein half-life determination with CON shRNA or FIEL1 shRNA expression (H). Data represent mean values ± SEM (n = 3 independent experiments; *, P < 0.05 compared with Empty or to Control, Student’s t test). (I–J) Immunoblots (I) showing levels of PIAS4, TGFBR1, TGFBR2, SMAD7, Smurf1, and V5 after FIEL1 expression. Protein quantification was graphed (J). Data represent mean values ± SEM (n = 3 independent experiments; *, P < 0.05 compared with 0 µg FIEL1, Student’s t test). (K) mRNA levels of PIAS4 upon FIEL1 expression was measured using two sets of PIAS4 RT-PCR primers. Data represent mean values ± SEM (n = 3 independent experiments). (L) MRC5 cells were treated with TGFβ in a time- or dose-dependent manner; cells were collected and immunoblotted for FIEL1 and PIAS4. Endogenous FIEL1 was also immunoprecipitated and immunoblotted for PIAS4 (n = 3). (M) PIAS4 and FIEL1 immunoblotting from lung tissues samples from five control and five IPF patients. PIAS4 and both the shorter and longer forms of KIAA0317 were quantified using ImageJ and graphed. Data represent mean values (n = 5 patients; NS, not significant *, P < 0.05 compared with CON, Student’s t test). (N) C57BL/6J mice were treated i.t. with bleomycin (0.02 U) for up to 21 d. Mice were then euthanized, and lungs were isolated and assayed for PIAS4 and FIEL1 immunoblotting. Bands corresponding to each protein on immunoblots were quantified using ImageJ software, and the results are displayed graphically. Data represent mean values ± SEM (n = 4–5 mice per group; *, P < 0.05 compared with day 0, Student’s t test).

FIEL1–PIAS4 pathway in pulmonary fibrosis. (A) PIAS4 protein half-life determination in MLE cells transfected with empty plasmid or ubiquitin plasmid (n = 2). (B) PIAS4 protein half-life determination with MG132 or leupeptin treatment (n = 3). (C) Immunoblots (top) showing levels of PIAS4 protein and V5 after KIAA0317 (823 aa, AREL1) and (789 aa, FIEL1) plasmid expression. PIAS4 protein quantification was normalized and graphed (bottom). Data represent mean values ± SEM (n = 3 independent experiments; *, P < 0.05 compared with 0 µg plasmid, Student’s t test). (D) PIAS4 protein was immunoprecipitated from cell lysate using a PIAS4 antibody and coupled to protein A/G beads. PIAS4 beads were then incubated with in vitro–synthesized products expressing HIS-V5-FIEL1 (789 aa) or HIS-V5-AREL1 (823 aa). After washing, proteins were eluted and processed for V5 immunoblotting (n = 2). (E) In vitro ubiquitination assay. Purified E1 and E2 components were incubated with V5-PIAS4 and FIEL1. The full complement of ubiquitination reaction components (second lane) showed polyubiquitinated PIAS4 proteins (n = 3). (F) Immunoblots showing levels of PIAS proteins and V5 after ectopic FIEL1 or UBE3B expression. (G and H) PIAS4protein half-life determination in MLE cells with empty plasmid or FIEL1 expression (G); PIAS4 protein half-life determination with CON shRNA or FIEL1 shRNA expression (H). Data represent mean values ± SEM (n = 3 independent experiments; *, P < 0.05 compared with Empty or to Control, Student’s t test). (I–J) Immunoblots (I) showing levels of PIAS4, TGFBR1, TGFBR2, SMAD7, Smurf1, and V5 after FIEL1 expression. Protein quantification was graphed (J). Data represent mean values ± SEM (n = 3 independent experiments; *, P < 0.05 compared with 0 µg FIEL1, Student’s t test). (K) mRNA levels of PIAS4 upon FIEL1 expression was measured using two sets of PIAS4 RT-PCR primers. Data represent mean values ± SEM (n = 3 independent experiments). (L) MRC5 cells were treated with TGFβ in a time- or dose-dependent manner; cells were collected and immunoblotted for FIEL1 and PIAS4. Endogenous FIEL1 was also immunoprecipitated and immunoblotted for PIAS4 (n = 3). (M) PIAS4 and FIEL1 immunoblotting from lung tissues samples from five control and five IPF patients. PIAS4 and both the shorter and longer forms of KIAA0317 were quantified using ImageJ and graphed. Data represent mean values (n = 5 patients; NS, not significant *, P < 0.05 compared with CON, Student’s t test). (N) C57BL/6J mice were treated i.t. with bleomycin (0.02 U) for up to 21 d. Mice were then euthanized, and lungs were isolated and assayed for PIAS4 and FIEL1 immunoblotting. Bands corresponding to each protein on immunoblots were quantified using ImageJ software, and the results are displayed graphically. Data represent mean values ± SEM (n = 4–5 mice per group; *, P < 0.05 compared with day 0, Student’s t test).

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