Figure 6.
LIR-dependent binding to ATG8s stimulates LMX1B-driven target gene transcription. (A) GFP-trap IP of wild-type and LIR mutant (Δ308-317) LMX1B-FLAG in lysates of HEK293T co-expressing GFP-ATG8 family members. 5% of protein lysate from equivalent GFP-expressing cells is shown as a representative of “input.” Immunoblotting for p62 is included as a positive control for binding. Molecular weight markers are shown in kD. (B) Immunofluorescence staining of ΔLIR mutant (Δ308-317) LMX1B-FLAG expressed in HEK293T cells. Bar = 20 µm. (C and D) LMX1B turnover in HEK293T stably expressing wild-type or Δ308-317 LMX1B-FLAG. Cells were treated with CHX (50 µg/ml; 16 h) in the absence or presence BafA1 (20 nM). Representative blot (C) and quantitation relative to GAPDH (D). Molecular weight markers are shown in kD. Mean ± SD (n = 3); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: ***P < 0.001. (E) LIR mutant Δ308-317 LMX1B fails to stimulate FLAT-luciferase activity. To the left, luciferase FLAT element sequences (top), and schematic of the proposed role of ATG8 proteins as LMX1B co-factors (bottom). To the right, quantitation of expression driven by wild-type and Δ308-317 LMX1B in HEK293T cells. Levels were normalized to empty vector control (pcDNA 3.1). Mean ± SD (n = 3–4); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test comparing scrambled sequence against NURR1, pro-insulin, TFEB, ULK1, UVRAG, and PINK1: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) LC3B siRNA silencing dampens LMX1B-mediated luciferase reporter expression. Example LC3B immunoblot (left, top) and scramble, NURR1, pro-insulin, ULK1, UVRAG, and PINK1 FLAT promoter luciferase assay (LightSwitch) driven by wild-type or Δ308-317 LMX1B-FLAG Levels are presented relative to empty vector control (pcDNA 3.1). Mean ± SD (n = 3–4); one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) GABARAP-L1 siRNA silencing dampens LMX1B-mediated luciferase reporter expression. Example GABARAP-L1 immunoblot (left, top) and scramble, NURR1, pro-insulin, ULK1, UVRAG, and PINK1 FLAT promoter luciferase assay (LightSwitch) driven by wild-type or Δ308-317 LMX1B-FLAG. Levels are presented relative to empty vector control (pcDNA 3.1). Mean ± SD (n = 3); one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

LIR-dependent binding to ATG8s stimulates LMX1B-driven target gene transcription. (A) GFP-trap IP of wild-type and LIR mutant (Δ308-317) LMX1B-FLAG in lysates of HEK293T co-expressing GFP-ATG8 family members. 5% of protein lysate from equivalent GFP-expressing cells is shown as a representative of “input.” Immunoblotting for p62 is included as a positive control for binding. Molecular weight markers are shown in kD. (B) Immunofluorescence staining of ΔLIR mutant (Δ308-317) LMX1B-FLAG expressed in HEK293T cells. Bar = 20 µm. (C and D) LMX1B turnover in HEK293T stably expressing wild-type or Δ308-317 LMX1B-FLAG. Cells were treated with CHX (50 µg/ml; 16 h) in the absence or presence BafA1 (20 nM). Representative blot (C) and quantitation relative to GAPDH (D). Molecular weight markers are shown in kD. Mean ± SD (n = 3); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: ***P < 0.001. (E) LIR mutant Δ308-317 LMX1B fails to stimulate FLAT-luciferase activity. To the left, luciferase FLAT element sequences (top), and schematic of the proposed role of ATG8 proteins as LMX1B co-factors (bottom). To the right, quantitation of expression driven by wild-type and Δ308-317 LMX1B in HEK293T cells. Levels were normalized to empty vector control (pcDNA 3.1). Mean ± SD (n = 3–4); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test comparing scrambled sequence against NURR1, pro-insulin, TFEB, ULK1, UVRAG, and PINK1: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) LC3B siRNA silencing dampens LMX1B-mediated luciferase reporter expression. Example LC3B immunoblot (left, top) and scramble, NURR1, pro-insulin, ULK1, UVRAG, and PINK1 FLAT promoter luciferase assay (LightSwitch) driven by wild-type or Δ308-317 LMX1B-FLAG Levels are presented relative to empty vector control (pcDNA 3.1). Mean ± SD (n = 3–4); one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) GABARAP-L1 siRNA silencing dampens LMX1B-mediated luciferase reporter expression. Example GABARAP-L1 immunoblot (left, top) and scramble, NURR1, pro-insulin, ULK1, UVRAG, and PINK1 FLAT promoter luciferase assay (LightSwitch) driven by wild-type or Δ308-317 LMX1B-FLAG. Levels are presented relative to empty vector control (pcDNA 3.1). Mean ± SD (n = 3); one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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