rabbit polyclonal anti tfe3  (Proteintech)

Journal: iScience

Article Title: Stimulation to secrete insulin induces pancreatic β-cell dysfunction through Tfe3 activation

doi: 10.1016/j.isci.2026.115312

Figure Lengend Snippet: Tfe3 is activated by stimuli to secrete insulin (A) Schematic representation of the long and short isoforms of Tfe3. Previously identified phosphoracceptor Ser/Thr residues are indicated. Rag-BD, Rag-binding domain; TAD, transactivation domain; bHLH-zip, basic-helix-loop-helix zipper domain. (B) Glucose stimulation induces de-phosphorylation of Tfe3. Extracts of MIN6 cells grown in the presence of 12.5 mM glucose or stimulated with 25 mM glucose for 6 h were analyzed by immunoblotting using an anti-Tfe3 antibody ( n = 3). Two Tfe3 isoforms and their phosphorylated and non-phosphorylated species are indicated. Representative blots of three independent experiments are shown. (C) KCl stimulation induces de-phosphorylation of Tfe3. Extracts of MIN6 cells treated with KCl for 1 h were analyzed by immunoblotting using an anti-Tfe3 antibody ( n = 3). (D) KCl stimulation of MIN6 cells induces nuclear accumulation of Tfe3. Cells treated with KCl (20 mM) and/or nimodipine (10 μM) were stained with an anti-Tfe3 antibody (top) and DAPI (bottom). Quantification of nuclear Tfe3 signal intensities in a representative experiment ( n = 2) is shown in a boxplots (right). In total, >788 cells were counted per condition per repeat. Scale bar, 10 μm. (E) KCl stimulation induces nuclear accumulation of Tfe3 in primary islet cells. Primary islets were treated with KCl (40 mM) and/or nimodipine (10 μM). Whole islets were stained with an anti-Tfe3 antibody (middle) and DAPI (bottom) and imaged using a confocal microscope. Top, shows merged images of Tfe3 and DAPI. Representative images from four independent experiments are shown. Scale bar, 20 μm. (F) Immunofluorescence staining of Tfe3 (red) in pancreas sections from a pair of mice (pair #1, identical to that shown in F) given water or 20% glucose-supplemented water for 18 days. The sections were also stained for insulin (green) together with nuclear counterstaining with DAPI (blue). Representative images of sections from the pair are shown ( n = 3). Sections from each pair were simultaneously processed for staining and images were acquired under identical settings. Nuclear Tfe3 signals in the pair of mice were quantified and are presented in violin and boxplots on the right. Cell numbers counted in each condition are also shown. Examination of two other pairs of samples yielded similar results ( C). Scale bar, 50 μm. ∗∗∗ p < 0.001; Kruskal-Wallis test followed by Steel-Dwass test (D) or Mann-Whitney U test (F). Boxes in (D) show median and lower and upper quantiles, and whiskers show 1.5 × interquartile range (IQR). The cross marks denote the averages. See also .

Article Snippet: Rabbit polyclonal anti-TFE3 (for ChIP and IF) , Proteintech , Cat# 14480-1-AP; RRID: AB_2199587.

Techniques: Binding Assay, De-Phosphorylation Assay, Western Blot, Staining, Microscopy, Immunofluorescence, MANN-WHITNEY

Journal: iScience

Article Title: Stimulation to secrete insulin induces pancreatic β-cell dysfunction through Tfe3 activation

doi: 10.1016/j.isci.2026.115312

Figure Lengend Snippet: KCl stimulation downregulates Mafa by activating Tfe3 (A–C) Enforced expression of Tfe3 suppresses endogenous Mafa levels. (A) Schematic structure of the expression plasmids and the Δ N1 mutant of Tfe3. EF1α, elongation factor 1α promoter; CMV, cytomegalovirus immediate-early enhancer and promoter. (B) MIN6 cells were transfected with the indicated plasmid and stained with anti-EGFP (green, on left) and anti-Mafa (red, on right) antibodies. Nuclei were counterstained with DAPI (blue). Arrows indicate EGFP-positive cells that received the plasmid. Mafa signal intensities in EGFP-negative and EGFP-positive cells in each condition in a representative experiment ( n = 3) were quantified and are presented in beeswarm and boxplots. Cell numbers counted are shown in the plots. Scale bar, 5 μm. (C) MIN6 cells were transfected with EGFP-Tfe3 Δ N1 expression plasmid and stained with anti-EGFP (green) and anti-Tfe3 (red) antibodies. Nuclei were counterstained with DAPI (blue). The rightmost panel shows a longer exposure image of the middle. Arrows and asterisks indicate EGFP-positive and EGFP-negative cells that received or did not receive the plasmid, respectively. Scale bar, 10 μm. (D–F) Knockdown of Tfe3 blunts KCl-stimulated downregulation of Mafa. (D) Schematic structure of the shRNA expression plasmids. (E) MIN6 cells transfected with the indicated plasmid were treated with 20 mM KCl for 6 h and subjected to immunofluorescence staining using anti-Tfe3 and anti-EGFP antibodies. The endogenous Tfe3 signal intensities were quantified and are presented in beeswarm and boxplots ( n = 2). Cell numbers counted in each condition are shown. (F) MIN6 cells transfected with the indicated plasmid were treated with 20 mM KCl for 6 h and subjected to immunofluorescence staining using anti-Mafa (red, top) and anti-EGFP (green, bottom) antibodies. Nuclei were stained with DAPI (blue). Arrows indicate EGFP-positive cells that were successfully transfected with the plasmids. Nuclear Mafa signal intensities in EGFP-negative and EGFP-positive cells were separately quantified and are presented in beeswarm and boxplots (right). Quantification of a representative experiment ( n = 2) and cell numbers counted in each condition are shown. Scale bar, 10 μm. ∗ p < 0.05; ∗∗∗ p < 0.001; n.s., not significant; Kruskal-Wallis test followed by Steel-Dwass test (B, E, and F).

Article Snippet: Rabbit polyclonal anti-TFE3 (for ChIP and IF) , Proteintech , Cat# 14480-1-AP; RRID: AB_2199587.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Staining, Knockdown, shRNA, Immunofluorescence

Journal: iScience

Article Title: Stimulation to secrete insulin induces pancreatic β-cell dysfunction through Tfe3 activation

doi: 10.1016/j.isci.2026.115312

Figure Lengend Snippet: Organelle stress and starvation activate Tfe3 and downregulate Mafa (A) Schematic view of the insulin biosynthesis and secretory pathway and sites of action of the organelle stressors used. (B) Tfe3 nuclear accumulation induced by stress-inducing reagents. MIN6 cells were treated with the indicated compounds (Cq, 20 mM; BfA, 20 μM; Mo, 50 nM; Tp, 100 nM; and Tu, 1 μM) for 6 h and stained with an anti-Tfe3 antibody (red, top) and DAPI (blue, bottom). Signal intensities of nuclear Tfe3 in a representative experiment ( n = 2) were quantified and are summarized in a boxplots (right). In total, >620 cells were counted per condition per repeat. Scale bar, 10 μm. (C) Organelle stresses reduce Mafa and Neurod1 levels in MIN6 insulinoma cells. Cells were treated with the indicated stressors for 6 h, and the cell extracts were analyzed by immunoblotting using the indicated antibodies. Quantification of the blot relative to βActin levels is indicated on the right (mean ± SEM; n = 3). (D) MIN6 cells grown in D-MEM supplemented with 12.5 mM glucose and 15% fetal bovine serum (Ctrl) were starved (0 mM glucose and 0% fetal bovine serum) for 24 h and stained with an anti-Tfe3 antibody (red, top). Tfe3 signal intensities in nuclei (blue) in a representative experiment ( n = 2) were quantified and are presented in a boxplot. In total, >567 cells were counted per condition per repeat. Scale bar, 20 μm. (E) Extracts from growing (Ctrl) or starved (Stv) MIN6 cells as in (D) were blotted with an anti-Mafa antibody. Quantification of the blot relative to βActin levels is indicated on the right (mean ± SEM; n = 3). (F) The mRNA levels of Mafa and Gapdh in MIN6 cells as in (D) were analyzed by quantitative RT-PCR (mean ± SEM; n = 3). (G and H) Immunostaining of pancreas sections from mice fed al libitum (Ctrl) or fasted for 16 h (Fasting) with the indicated antibodies. Images are representative of two independent experiments. Sections from each pair of mice were simultaneously processed for staining and images were acquired under identical settings. Quantification of nuclear Tfe3 (G) and Mafa (H) intensities in a pair of mice is indicated in violin plots on the right. Cell numbers counted are shown in the plots. Examination of another pairs of samples yielded similar results ( C and S5D). Scale bars, 50 μm. ∗ p < 0.05; ∗∗∗ p < 0.001; n.s., not significant; one-way ANOVA followed by Tukey’s multiple comparison test (B and C), Mann-Whitney U test (D, G, and H), or Student’s t test (E and F). See also .

Article Snippet: Rabbit polyclonal anti-TFE3 (for ChIP and IF) , Proteintech , Cat# 14480-1-AP; RRID: AB_2199587.

Techniques: Staining, Western Blot, Quantitative RT-PCR, Immunostaining, Comparison, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Type III Intermediate Filament Protein Peripherin Regulates Lysosomal Degradation Activity and Autophagy

doi: 10.3390/ijms26020549

Figure Lengend Snippet: Peripherin silencing affects lysosomal functionality. ( A ) Lysates of peripherin-silenced or control Neuro2a cells, as indicated, were subjected to Western blot analysis using antibodies against LAMP1, RAB7A, beclin 1, RILP, V1G1, peripherin, and Hsp90. Relative protein abundance was quantified by densitometric analysis normalizing against Hsp90. ( B ) Peripherin-silenced or control Neuro2A cells, as indicated, were subjected to immunofluorescence analysis using anti-TFEB antibody followed by Alexa488-conjugated secondary antibody, while nuclei were stained with DAPI. Bar = 10 µM. TFEB CTCF was calculated using ImageJ software. To check silencing efficiency, Western blot analysis was performed on same samples using antibodies against peripherin and Hsp90 as loading control. Samples of control and silenced cells were also subjected to Western blot analysis using antibodies against TFEB and TFE3. Relative protein abundance was quantified by densitometric analysis normalizing against Hsp90. ( C ) Lysates of control or peripherin-depleted Neuro2a cells were subjected to Western blot analysis using antibodies against RAB4, RAB5, RAB9, RAB11, peripherin, or Hsp90 as loading control. Relative protein abundance was quantified by densitometric analysis normalizing against Hsp90. ( D ) Peripherin-silenced or control Neuro2A cells were incubated with DQ-Red BSA for 8 h and then fixed and immunolabeled using anti-peripherin antibody followed by Alexa488-conjugated secondary antibody. Nuclei were stained with DAPI. Bar = 10 µM. DQ-BSA CTCF was calculated using ImageJ. ( E ) Peripherin-silenced or control Neuro2A cells were treated with cycloheximide for 1 h and then stimulated with EGF for 15 min and 3 h. Lysates were then subjected to Western blot analysis using antibodies against EGFR, peripherin, and β-actin as loading control. Percentage of degraded EGFR was calculated. ( F ) Lysates of control or peripherin-silenced Neuro2a cells were subjected to Western blot analysis using antibodies against p62, V0D1, peripherin, or Hsp90 as loading control. Relative protein abundance was quantified by densitometric analysis normalizing against Hsp90. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Article Snippet: Primary antibodies used in this study were goat polyclonal anti-peripherin (sc-7604, 1:500), mouse monoclonal anti-GAPDH (sc-25778, 1:2000), mouse monoclonal anti-RAB7A (sc-376362, 1:500), goat polyclonal anti-beclin 1 (sc-10086; 1:200), mouse monoclonal anti-Hsp90 (sc-13119, 1:5000), mouse monoclonal anti-MAPK1 (sc-514302; 1:500), mouse monoclonal anti-ATP6V0D1 (sc-81887, 1:100), mouse monoclonal anti-myc (sc-40, 1:50 for IF), rabbit polyclonal anti-myc (sc-789, 1:50 for IF), and mouse monoclonal anti-β-actin (sc-47778, 1:5000), which were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-TFE3 (HPA023881; 1:1000) obtained from ThermoFisher Scientific (Waltham, MS, USA); rabbit polyclonal anti-LAMP1 (ab24170, 1:4000), mouse monoclonal anti-V1G1 (sc25333, 1:100), and rabbit polyclonal anti-RILP (ab140188, 1:500) from Abcam (Cambridge, UK); rabbit polyclonal anti-peripherin (AB1530, 1:100 for IF) from Merck Millipore (Burlington, MS, USA); sheep polyclonal anti-EGFR (20-ES04, 1:1000) from Fitzgerald (North Acton, MS, USA); mouse monoclonal anti-LC3B (0231-1000, 1:500 for WB, 1:200 for IF) from Nanotools (Teningen, Germany); rabbit polyclonal anti-TFEB (A303-673A, 1:400) from Bethyl Laboratories (Montgomery, TX, USA); mouse monoclonal anti-LAMP1 (1D4B, 1:50 for IF) from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA); rabbit polyclonal anti-caspase 3 (9662, 1:1000), mouse monoclonal anti-caspase 9 (9508, 1:1000), rabbit polyclonal anti-RAB4 (2167, 1:1000), rabbit polyclonal anti-RAB5 (3547, 1:1000), rabbit polyclonal anti-RAB9 (5118, 1:1000), and rabbit polyclonal anti-RAB11 (5589, 1:2000) from Cell Signaling Technology (Danvers, MA, USA); and mouse monoclonal anti-SQSTM1/p62 (1:1000; 610833) from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Control, Western Blot, Immunofluorescence, Staining, Software, Incubation, Immunolabeling