Fig. 3: UCHL3 binds to and deubiquitinates p300 in response to TGF-β signaling. | Experimental & Molecular Medicine

Fig. 3: UCHL3 binds to and deubiquitinates p300 in response to TGF-β signaling.

From: The deubiquitinase UCHL3 mediates p300-dependent chemokine signaling in alveolar type II cells to promote pulmonary fibrosis

Fig. 3

a Quantification of p300 protein values normalized to β-actin levels. DUB-IN-2, mitoxantrone, PR-619, and P5091 were administered at a concentration of 0.05 μM. Degrasyn, GSK2643943A, and USP30 inhibitor 18 were administered at a concentration of 0.1 μM. LDN-57444, ML364, vialinin A, and AZ1 were administered at a concentration of 1 μM. b-AP15, TCID, spautin-1, IU1-47, ML323, and FT671 were administered at a concentration of 10 μM. b RLE-6TN cells were transfected with the indicated constructs, and an immunoprecipitation assay was performed with FLAG or HA antibodies. c The lysates of RLE-6TN cells were immunoprecipitated with UCHL3 or p300 antibodies and immunoblotted with the indicated antibodies. d Human lung samples were stained with the indicated antibodies; healthy individuals (n = 7), IPF patients (n = 14). Arrowheads indicate colocalized regions in ATII cells. Scale bar = 50 μm. The weighted colocalization efficiency was calculated using ZEN 3.0 software. e RLE-6TN cells were cotransfected with the indicated plasmids. MG132 (10 μM) was added for the final 6 h. Equal amounts of protein were immunoprecipitated with HA antibodies and immunoblotted with the indicated antibodies. f RLE-6TN cells were cotransfected with His-ubiquitin and HA-p300 plasmids. Cells were treated 24 h posttransfection with TCID (5 μM), and whole cell lysates were prepared. g RLE-6TN cells were treated with TCID for 24 hr followed by treatment with 20 mg/ml cycloheximide (CHX). p300 protein levels were normalized to β-actin. h RLE-6TN cells were transfected with empty, UCHL3, or UCHL3C95A expression vectors. Two days after transfection, the cells were treated with CHX for the indicated times. The levels of p300 protein were normalized to the level of β-actin. i Relative expression of Ccl2, Ccl7, Ccl12 mRNA levels in TCID (5 µM) treated RLE-6TN. Statistical analysis was performed with one-way ANOVA with Tukey’s test (a,i), the Mann‒Whitney test (d) or two-way ANOVA with Sidak’s test (g, h). Error bars represent the mean ± s.e.m. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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