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. Author manuscript; available in PMC: 2021 Nov 1.
Published in final edited form as: Cell Immunol. 2020 Sep 2;357:104203. doi: 10.1016/j.cellimm.2020.104203

Fig. 1.

Fig. 1.

Smad3 phosphorylation is induced by FLIL33 overexpression in primary fibroblasts. A. NHLFs from two different healthy donors (lanes 1, 2 and 3, 4) were electroporated with FLIL33-encoding (lanes 2, 4) or a control NULL (lanes 1, 3) plasmids, as indicated. After 48 h, cells were lysed, and western blotting analyses were performed with anti-phospho-Smad2/3 antibody. The membranes were then stripped and re-developed for total Smad2/3 and GAPDH as indicated. Note the pronounced Smad3 phosphorylation induced by FLIL33 overexpression. The FLIL33 overexpression-induced increases in Smad3 phosphorylation were statistically significant (Suppl. Fig. 1). Such stimulation of Smad3 phosphorylation by FLIL33 overexpression was consistent in all NHLFs tested on at least two, and in some cases more than ten, independent occasions in each of the three previously described [12] and additional five (Suppl. Fig. 1) studied primary cultures. B. FLIL33 overexpression activates Smad3 phosphorylation in a time-dependent fashion. NULL-transfected and FLIL33-overexpressing NHLFs were analyzed at 24 h, 48 h, and 72 h, as indicated. C. Only FLIL33 overexpression but not similar overexpression of MIL33 (aa 112–270), FLIL37, or MIL37 (aa 46–218) induces Smad3 phosphorylation. Western blots were developed for phospho-Smad2/3; the membrane was serially stripped and re-developed with the subsequent antibodies in the order shown for total Smad2/3, GAPDH, and HA (the C-terminal tag in all constructs), as indicated. D. The effect of FLIL33 overexpression on Smad2/3 phosphorylation is pronounced in primary NHLF and fibroblast from patients with IPF, but not observed in NIH3T3, HEK293, A549 cells, or SAEC. E. Phosphorylation of ERK1/2 is not affected by FLIL33 overexpression but is induced by TGF-β. F. FLIL33 overexpression has a limited impact on the levels of Smad4 and Smad7. NHLFs from two different healthy donors (lanes 1, 2 and 3, 4) were electroporated with FLIL33-encoding (lanes 2, 4) or a control NULL (lanes 1, 3) plasmids, as indicated. After 48 h, cells were lysed, and western blotting analyses were performed with anti-Smad4 antibody. The membranes were then stripped and re-developed for total Smad7 and GAPDH as indicated.