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. 2022 Dec 8;207(8):1042–1054. doi: 10.1164/rccm.202207-1332OC

Figure 4.

Figure 4.

TanFe (transcellular activator of nuclear FOXF1 expression) inhibits FOXF1 (Forkhead Box F1) ubiquitination. (A) TanFe decreases ubiquitination of the FOXF1 protein. IP was performed in protein extracts from Mouse Fetal Lung Mesenchyme-91 U (MFLM-91U) cells stably expressing His-Flag–tagged FOXF1 (HF-FOXF1) using the FLAG antibody followed by Western blot with ubiquitin antibodies. TanFe treatment (20 μM) was performed for 24 hours. Equal protein loading is shown by Western blot for FOXF1 (n = 3 pooled cell cultures). (B) Chromatin fractionation shows that TanFe increases FOXF1 amounts in the chromatin fraction. TanFe does not change HECTD1 amounts in cell fractions. Chromatin fractionation was performed in MFLM-91 U cells treated with 20 μM of TanFe for 24 hours. Cell lysates were pooled from three independent cell cultures. (C) Western blot shows that inhibition of HECTD1 by siRNA (siHECTD1) increases FOXF1 protein amounts in MFLM-91 U cells. siCtrl is scrambled siRNA (n = 3 pooled cell cultures). (D) Overexpression of HECTD1 decreases FOXF1 expression. Cells were transfected with the HA-HECTD1 expression vector, and the Western blot was performed 48 hours after transfection (n = 3 pooled cell cultures). Chrom = chromatin; Ctrl = control; Cyto-Nucleo = cytoplasmic and nuclear extract; IB = Immunoblot; IP = immunoprecipitation.