Journal: The Journal of Biological Chemistry

Article Title: Antibody validation for Western blot: By the user, for the user

doi: 10.1074/jbc.RA119.010472

Figure Lengend Snippet: Validation of IDH1 antibody using purified recombinant protein in multicolor and chemiluminescent Western blotting. Multicolor and chemiluminescent Western blottings were performed using 10% Bis-Tris SDS-polyacrylamide gel and MOPS buffer system to validate the IDH1 antibody using a purified recombinant IDH1 protein (0.16 μg) containing a c-Myc tag in addition to HEK293T and HeLa whole-cell lysates. A, c-Myc protein tag present on the purified IDH1 recombinant protein is detected in the 700-nm channel ( red ) at 50 kDa via mouse anti-c-Myc antibody (ab32;1 μg/ml) using IRDye 680RD goat anti-mouse IgG (H + L) for detection. Some overspill of the recombinant protein into neighboring lanes is observed ( white box ). B, IDH1 recombinant protein and endogenous IDH1 protein, present in HEK293T and HeLa, is detected in the 800-nm channel ( green ) at 55 and 50 kDa, respectively, using rabbit anti-IDH1 antibody (ab172964; 1.2 μg/ml) and IRDye 800CW goat anti-mouse IgG (H + L) for detection. C, when both 700- and 800-nm channels are displayed, the signal from ab32 and ab172964 overlaps at 50 kDa, identifying the c-Myc–tagged IDH1 protein. No overlap is seen for the endogenous IDH1 present in HEK293T and HeLa whole-cell lysates. A–C , lysates loaded per lane are as follows: 20 μg of blocking buffer: Odyssey blocking buffer (TBS); imager: Odyssey® CLx; resolution: 169 μm; intensity: auto mode. Chameleon TM Duo pre-stained protein ladder for accurate sizing of protein bands. D, single blot was split into two halves ( green line ) to be incubated with either rabbit anti-IDH1 antibody (ab172964; 0.115 μg/ml) or the corresponding rabbit monoclonal IgG isotype control (ab172730; 0.166 μg/ml) to detect the endogenous IDH1 protein present in HeLa and HEK293T as well IDH1 recombinant protein. Both halves were incubated with HRP-conjugated goat anti-mouse IgG (H + L). E, single blot was split into two halves ( green line ) to be incubated with either mouse anti-c-Myc antibody (ab32; 1 μg/ml) or the corresponding mouse monoclonal IgG1 isotype control (ab18443; 1 μg/ml) to detect c-Myc protein tag present on the purified IDH1 recombinant protein but absent in HEK293T and HeLa whole-cell lysates. Both halves were incubated with HRP-conjugated goat anti-rabbit IgG (H + L). Blots were detected with WesternSure® PREMIUM chemiluminescent substrate (LI-COR 926–95000) and imaged on an Odyssey® Fc with the following resolution: 125 μm and exposure of 2 min. Lysate loaded per lane: 20 μg; protein ladder: WesternSure® pre-stained chemiluminescent protein ladder (LI-COR 926-980000); blocking buffer: intercept blocking buffer (TBS); intercept T20 (TBS) antibody diluent.

Article Snippet: Isocitrate dehydrogenase (IDH1) (NM_005896) human recombinant protein (OriGene no. TP310582), IDH1 (NM_005896) human overexpression lysate supplied with parental HEK293T lysate (OriGene no. LY401782; HEK293T LY500001; lot no. 0076CF), and IDH1 knockout cell lysate (supplied with parental HeLa control lysate) (Origene no. LC810112, LC810Hela; lot no. 1601) were mixed with either 2× protein loading buffer (PLB) (LICOR no. 928-40004) or 2× SDS buffer (OriGene) and denatured by boiling at 97 °C for 5 min.

Techniques: Biomarker Discovery, Purification, Recombinant, Western Blot, Blocking Assay, Staining, Incubation, Control

Journal: EMBO Reports

Article Title: VGLL4 modulates Paneth cells and sustains intestinal homeostasis

doi: 10.1038/s44319-026-00699-3

Figure Lengend Snippet: ( A ) IF staining of small intestinal samples from Control and ISC-specific knockout (ISC-KO) mice at 1 week after TAM injection. Top 2 panels: Staining for Ki67 (gray), GFP (green), RFP (purple) and DAPI (blue). Bottom 2 panels: Staining for Lysozyme (gray), GFP (green), RFP (purple) and DAPI (blue). Scale bars, 10 μm. ( B ) Quantification of Ki67 + cells and Lysozyme + cells per crypt in ( A ) ( n = 4). Unpaired Student’s t test. ns P = 0.5512. ** P = 0.0281. ( C ) qRT-PCR analysis of Gfi1 and Sox9 mRNA levels in Vgll4 fl/fl and Vgll4 IEC-KO mice ileum samples ( n = 5). Unpaired Student’s t test. *** P = 0.0008. ns P = 0.1057. ( D ) Immunoblot analysis of GFI1, SOX9, ATOH1, and VGLL4 in ileum samples of Vgll4 fl/fl and Vgll4 IEC-KO mice. Samples were derived from the same experiment and blots were processed in parallel. ( E ) Quantification of protein levels in ( D ) ( n = 5). Unpaired Student’s t test. GFI1: ** P = 0.0018. ATOH1: ns P = 0.6427. SOX9: ns P = 0.4877. ( F ) IF staining of GFI1 (red) in Vgll4 fl/fl and Vgll4 IEC-KO mice small intestinal organoids ( n = 5). DAPI, blue. Scale bars, 30 μm. ( G ) Co-immunoprecipitation assay results between ATOH1 and TEAD4 in HEK293T cells. ( H ) Co-immunoprecipitation assay results between ATOH1 and TEAD4 in HEK293T cells with DNase. ( I ) Co-immunoprecipitation assay results between ATOH1 and different mutations of TEAD4 in HEK293T cells. ( J ) Co-immunoprecipitation assay results between ATOH1 and VGLL4 in HEK293T cells with or without TEAD4. ( K ) Immunoblot analysis of GST pull-down assay between purified ATOH1-ΔN, VGLL4, and TEAD4 proteins. Results are shown as mean + SD. ns no significance. .

Article Snippet: HEK293T , ATCC , ACS-4500.

Techniques: Staining, Control, Knock-Out, Injection, Quantitative RT-PCR, Western Blot, Derivative Assay, Co-Immunoprecipitation Assay, Pull Down Assay, Purification

Journal: EMBO Reports

Article Title: VGLL4 modulates Paneth cells and sustains intestinal homeostasis

doi: 10.1038/s44319-026-00699-3

Figure Lengend Snippet: ( A ) qRT-PCR analysis of Math1 from Vgll4 fl/fl and Vgll4 IEC-KO mice ileum samples ( n = 5). Unpaired Student’s t test. ns P = 0.7009. ( B ) Co-immunoprecipitation assay results between ATOH1 and VGLL4 in HEK293T cells. ( C ) Schematic illustration of TEAD4 and its short forms. ( D ) Co-immunoprecipitation assay results between ATOH1 and TEAD4 short forms in HEK293T cells. The black arrow indicates IgG heavy chain. ( E ) Co-immunoprecipitation assay results between ATOH1 and TEAD4-N and TEA domain short forms in HEK293T cells. ( F ) Schematic illustration of ATOH1 and its short forms. ( G ) Co-immunoprecipitation assay results between ATOH1 short forms and TEAD4 in HEK293T cells. ( H ) Coomassie brilliant blue staining of the GST-pulldown assay between ATOH1-ΔN, VGLL4, and TEAD4. ( I ) FLAG antibody pulldown assay results between VGLL4-TEAD4-ATOH1 complex in HEK293T cells overexpressing FLAG-VGLL4, MYC-ATOH1 and HA-TEAD4. ( J ) ChIP-qPCR analysis of TEAD4 enrichment at the GFI1 promoter in HEK293T cells overexpressing FLAG-TEAD4. Unpaired Student’s t test. **** P < 0.0001. Three biological replicates per group. ( K ) ChIP-qPCR analysis of TEAD4 at the GFI1 promoter in 293T cells with or without VGLL4 overexpression. Unpaired Student’s t test. **** P < 0.0001. Left *** P = 0.0007. Right *** P = 0.0003. Three biological replicates per group. ( L ) ChIP-qPCR analysis of ATOH1 at the GFI1 promoter in HEK293T cells with or without VGLL4 overexpression. Unpaired Student’s t test. Left *** P = 0.0003. Right *** P = 0.0007. Three biological replicates per group. ( M ) A model of GFI1 luciferase construction strategy. Red arrow: TBS, TEAD4 binding sequence. Black arrow: ABS, ATOH1 binding sequence. RR: regulatory region. ( N ) GFI1 Luc reporter activity in HCoEpiC cells transfected with ATOH1, VGLL4, and TEAD4. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. ( O ) GFI1 Luc reporter activity in HCoEpiC cells transfected with ATOH1, TEAD4 wild-type or mutant form, VGLL4 wild-type or mutant form. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. Results are shown as mean + SD. ns no significance.

Article Snippet: HEK293T , ATCC , ACS-4500.

Techniques: Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, GST Pulldown Assay, ChIP-qPCR, Over Expression, Luciferase, Binding Assay, Sequencing, Activity Assay, Transfection, Mutagenesis

Journal: EMBO Reports

Article Title: VGLL4 modulates Paneth cells and sustains intestinal homeostasis

doi: 10.1038/s44319-026-00699-3

Figure Lengend Snippet: ( A ) Heatmap analysis of defensin expression with RNA-seq data ( n = 4). ( B ) ChIP-qPCR analysis of TCF4 enrichment at the DEFA5 promoter in SW620 cells. Unpaired Student’s t test. *** P = 0.0008. Three biological replicates per group. ( C ) ChIP-qPCR analysis of TEAD4 enrichment at the DEFA5 promoter in SW620 cells. Unpaired Student’s t test. *** P = 0.0003. Three biological replicates per group. ( D ) Luciferase reporter activity driven by DEFA5 promoter in HEK293T cells transfected with indicated plasmids. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. ( E ) Luciferase reporter activity driven by DEFA5 promoter in HEK293T cells transfected with TCF4, VGLL4 and TEAD4 wild-type or TEAD4-TEA short form. One-way ANOVA. **** P < 0.0001. *** P = 0.0004. Three biological replicates per group. ( F ) Luciferase reporter activity driven by DEFA5 promoter in HEK293T cells transfected with TCF4, TEAD4 and VGLL4 wild-type or VGLL4-HF4A short form. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. ( G ) Luciferase reporter activity driven by DEFA5 promoter in HCoEpiC cells transfected with indicated plasmids. One-way ANOVA. **** P < 0.0001. ** P = 0.0025. Three biological replicates per group. ( H ) Luciferase reporter activity driven by DEFA5 promoter in HCoEpiC cells transfected with TCF4, TEAD4 wild-type or TEAD4-TEA short form, and VGLL4 wild-type or VGLL4-HF4A short form. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. Results are shown as mean + SD.

Article Snippet: HEK293T , ATCC , ACS-4500.

Techniques: Expressing, RNA Sequencing, ChIP-qPCR, Luciferase, Activity Assay, Transfection

Journal: Scientific reports

Article Title: TSC complex decrease the expression of mTOR by regulated miR-199b-3p.

doi: 10.1038/s41598-025-85706-8

Figure Lengend Snippet: Fig. 4. miR-199b-3p regulate the expression of mTOR (A) Quantitative analysis of mTOR in fibroblasts using qRT-PCR. (B) Quantitative analysis of mTOR in fibroblasts using western blot and the statistics. The originals gels can be seen in the supplementary figure S5. (C) Predicted miR-199b-3p target sequence of the mTOR 3’UTR using miRBase. (D) Quantification of miR-199b-3p and mRNA using qRT-PCR, the WT represents the mixture of the equal amount total RNA from the two healthy fibroblasts. Relative expression levels were normalized with GAPDH. (E). Luciferase reporter assay. (F) Quantitative analysis of mTOR in HEK293T cells at 48 h post-treatment with miR-199b-3p mimics (low panel) and the statistics (up panel). The originals gels can be seen in the supplementary figure S5. (G) Western bolt analysis of p-AKT, AKT, p-p70S6K, p70S6K in HEK293T cells at 48 h post-treatment with miR-199b-3p mimics,β-actin served as a control of total protein. (H) Quantitative analysis of mTOR in HEK293T cells at 48 h post-treatment with the inhibitor specific to miR- 199b-3p (low panel) and the statistics (up panel). (I) Western bolt analysis of p-AKT, AKT, p-p70S6K, p70S6K in HEK293T cells at 48 h post-treatment with the inhibitor specific to miR-199b-3p, β-actin served as a control of total protein. The originals gels can be seen in the supplementary figure S5.

Article Snippet: HEK293T cells were maintained in DMEM (Boster) supplemented with 10% FBS (BI).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Sequencing, Luciferase, Reporter Assay, Control

Journal: Scientific reports

Article Title: TSC complex decrease the expression of mTOR by regulated miR-199b-3p.

doi: 10.1038/s41598-025-85706-8

Figure Lengend Snippet: Fig. 5. TSC complex regulates the endogenous content of miR199b-3p, *p < 0.05, **p < 0.01,***p < 0.001 comparison with WT. (A) The efficiency of knockdown TSC1 in shTSC1-HEK293T cell line. The originals gels can be seen in the supplementary figure S6. (B) The expression of miR-199b-3p in shTSC1-HEK293T cells and overexpression TSC1-WT or TSC1-N837fs in HEK293T cell line. (C) Quantitative analysis of mTOR in shTSC1-HEK293T cell line and overexpression TSC1-WT or TSC1 N837fs in HEK293T cell line using western blot (low panel) and the statistics (up panel). The originals gels can be seen in the supplementary figure S6. (D) The efficiency of knockdown TSC2 in shTSC2-HEK293T cell line. The originals gels can be seen in the supplementary figure S6. (E) The expression of miR-199b-3p in shTSC2-HEK293T cell line and overexpression TSC2-WT or TSC2- Q371fs in HEK293T cell line. (F) Quantitative analysis of mTOR in shTSC2-HEK293T cell line (low panel) and overexpression TSC2-WT or TSC2- Q371fs in HEK293T cell line using Western blot (low panel) and the statistics analysis (up panel). The originals gels can be seen in the supplementary figure S6.

Article Snippet: HEK293T cells were maintained in DMEM (Boster) supplemented with 10% FBS (BI).

Techniques: Comparison, Knockdown, Expressing, Over Expression, Western Blot