Journal: Nature Communications

Article Title: TFEB degradation is regulated by an IKK/β-TrCP2 phosphorylation-ubiquitination cascade

doi: 10.1038/s41467-026-71001-1

Figure Lengend Snippet: a Immunoblot analysis of HeLa/TFEB and HeLa/TFEB-6xA using the indicated antibodies (n  =  3 independent replicates for each group). b Quantification of the immunoblots in ( a ), normalized to corresponding GAPDH levels. c Relative TFEB-3xFlag mRNA levels determined by RT qPCR in samples shown in panel ( a ). The expression levels were normalized to the housekeeping gene, GAPDH . d HeLa/TFEB and HeLa/TFEB-6xA were treated with 100 μg/ml cycloheximide (CHX), and the cells were harvested at the indicated time points. Immunoblot analyses were performed using the indicated antibodies ( n =  3 independent replicates for each group). To facilitate quantification, the ratio between the concentrations of loaded proteins for WT TFEB and TFEB-6 x A is 5:1 to take into account that TFEB-6 x A’ signal intensity is several-fold greater than that of WT TFEB. e Quantification of the immunoblots in ( d ), normalized to corresponding β-actin levels. f HeLa/TFEB and HeLa/TFEB-6 x A were treated with 20 ng/ml TNFα, and the cells were harvested at the indicated time points. Immunoblot analyses were performed using the indicated antibodies ( n =  3 independent replicates for each group). g Quantification of the immunoblots in ( f ), normalized to corresponding GAPDH levels. h HEK293T cells were cotransfected with TFEB-3 x Flag, HA-ubiquitin, and one of the indicated E3 ligases (β-TrCP1, β-TrCP2, STUB1 or DCAF7). 24 h after transfection, TFEB proteins were immunoprecipitated, and the ubiquitination levels were checked using the HA antibody. i HEK293T cells were cotransfected with HA-ubiquitin and TFEB-3xFlag. 24 h after transfection, the cells were split and subjected to a second round of transfection with scramble or FBXW11 (β-TrCP2) siRNA. After an additional 28 h, the cells were incubated with 100 nM CFZ or DMSO for 20 h before harvest. TFEB proteins were then immunoprecipitated, and ubiquitination levels were assessed using an HA antibody. h , i Representative images are shown from n = 3 independent experiments with similar results. Results are presented as mean ± SEM. p- values are calculated based on two-sided student’s t test. Source data are provided as a Source Data file.

Article Snippet: TFEB_6 x A-3 × Flag and TFEB_6xE-3 × Flag were synthesized by Twist Bioscience and cloned into a pcDNA3.1-based expression vector using In-Fusion® Snap Assembly Master Mix (Cat# 638948, Takara Bio).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Incubation

Journal: Nature Communications

Article Title: TFEB degradation is regulated by an IKK/β-TrCP2 phosphorylation-ubiquitination cascade

doi: 10.1038/s41467-026-71001-1

Figure Lengend Snippet: a Immunoblot analysis of HEK293T cells transiently transfected with scramble or FBXW11 siRNA ( n =  3 independent experiments) using the indicated antibodies. b Quantification of the immunoblots in ( a ), normalized to corresponding β-actin levels. c Relative FBXW11 mRNA levels determined by RT-qPCR in samples shown in panel ( a ). d Relative TFEB mRNA levels determined by RT-qPCR in samples shown in panel ( a ). e Immunoblot analysis of WT MEFs and MEF lines bearing targeted deletion of the genes encoding IKKα, IKKβ, or IKKγ, transiently transfected with scramble or Fbxw11 siRNA ( n =  4 independent experiments) using the indicated antibodies. f Quantification of the immunoblots in ( e ), normalized to corresponding GAPDH levels. g Relative Fbxw11 mRNA levels determined by RT-qPCR in samples shown in panel ( e ) ( n =  3 technical replicates). h Immunoblot analysis of HeLa/TFEB and HeLa/TFEB-6xA transiently transfected with scramble or FBXW11 siRNA ( n =  3 independent experiments) using the indicated antibodies. To facilitate quantification, the ratio between the concentrations of loaded proteins for WT TFEB and TFEB-6 x A is 5:1 to take into account that TFEB-6 x A’ signal intensity is several-fold greater than that of WT TFEB. i Quantification of the immunoblots in ( h ), normalized to corresponding β-actin levels. j Relative FBXW11 mRNA levels determined by RT-qPCR in samples shown in panel ( h ). k HEK293T cells were cotransfected with HA-ubiquitin and TFEB-3 x Flag or TFEB 6xA-3 x Flag. 24 h after transfection, the cells were split and subjected to a second round of transfection with scramble or FBXW11 siRNA. After an additional 48 h, TFEB proteins (WT and 6xA) were immunoprecipitated, and ubiquitination levels were assessed using an HA antibody. Representative images are shown from n = 3 independent experiments with similar results. l HeLa/TFEB were transiently transfected with scramble or FBXW11 siRNA. 48 h later, the cells were treated with 20 ng/ml TNFα for 15 minutes. Immunoblot analyses were performed using the indicated antibodies ( n =  3 independent replicates for each group). m Quantification of the immunoblots in ( l ), normalized to corresponding β-actin levels. n HEK293T cells were transiently transfected with scramble or FBXW11 siRNA. 72 h later, the cells were treated with 250 nM IKK-16 or DMSO for 6 h. Immunoblot analyses were performed using the indicated antibodies ( n =  3 independent replicates for each group). o Quantification of the immunoblots in ( n ), normalized to corresponding β-actin levels. Results are presented as mean ± SEM. p- values are calculated based on two-sided student’s t test. Source data are provided as a Source Data file.

Article Snippet: TFEB_6 x A-3 × Flag and TFEB_6xE-3 × Flag were synthesized by Twist Bioscience and cloned into a pcDNA3.1-based expression vector using In-Fusion® Snap Assembly Master Mix (Cat# 638948, Takara Bio).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Ubiquitin Proteomics, Immunoprecipitation

Journal: Nature Communications

Article Title: TFEB degradation is regulated by an IKK/β-TrCP2 phosphorylation-ubiquitination cascade

doi: 10.1038/s41467-026-71001-1

Figure Lengend Snippet: a HeLa/TFEB, HeLa/TFEB-6xA and HeLa/TFEB-KKRR were treated with DMSO, Torin1, or starved for amino acids (HBSS) for 4 hours prior to immunofluorescent labeling of TFEB with Flag antibody. Representative images are shown. Scale bar: 30 μm. b Quantification of TFEB NTI of cells under the above-mentioned three conditions in ( a ). n = 30 cells per condition. One-way ANOVA with Tukey’s multiple comparisons test. c HeLa/TFEB, HeLa/TFEB-6xA and HeLa/TFEB-KKRR treated as in ( a ) were fractionated into cytosol and nuclei. Immunoblot analyses of each fraction was performed with the indicated antibodies. Representative images are shown from n = 3 independent experiments with similar results. d HEK293T cells were transfected with 3xFlag tagged WT TFEB, TFEB-6 x A or TFEB-KKRR. 24 hours after transfection, immunoblot analyses were performed with the indicated antibodies ( n =  3 independent experiments) (upper); quantification of TFEB-6xA and KKRR levels, respectively, relative to WT TFEB levels (lower). Two-sided student’s t test. e Expression analysis of cells in ( d ). Gene expression levels were normalized to the housekeeping gene, GAPDH. MCOLN1, ATP6V1H, GNS and LAMP1 ( n = 3 per group); SCPEP1, NEU1, CTSA and PIP4P1 (pcDNA, n = 3; WT/6xA/KKRR, n = 4). Multiple two-sided t tests with Benjamini–Hochberg FDR correction. f HEK293T cells were transfected with 3 x Flag tagged WT TFEB, TFEB-6 x A or TFEB-KKRR, together with V5 tagged full length human tau with P301L mutation. 48 h after transfection, immunoblot analyses were performed with the indicated antibodies ( n =  6 independent replicates for each group). g Quantification of total tau, phospho-tau and TFEB levels, respectively. One-way ANOVA with Tukey’s multiple comparisons test. Results are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: TFEB_6 x A-3 × Flag and TFEB_6xE-3 × Flag were synthesized by Twist Bioscience and cloned into a pcDNA3.1-based expression vector using In-Fusion® Snap Assembly Master Mix (Cat# 638948, Takara Bio).

Techniques: Labeling, Western Blot, Transfection, Expressing, Gene Expression, Mutagenesis