Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Cardioprotective efficacy of Xin-shu-bao tablet in heart failure with reduced ejection fraction by modulating THBD/ARRB1/FGF1/STIM1 signaling.

doi: 10.1016/j.biopha.2023.115119

Figure Lengend Snippet: Fig. 7. Effect of XSB on the expression of the target proteins in the MI-induced pro- and post- HFrEF mice. Western blot analysis of the proteins selected from differentially expressed proteins. The intensities of the bands from the Western blots were quantified and normalized to the corresponding intensity of the GAPDH band. (A) Expression levels of the THBD and FGF1 proteins after 8 weeks of XSB administration; expression levels of PTBP1 and STIM1 proteins after 6 weeks of XSB administration; expression levels of ARRB1 and HIP1 proteins after 4 weeks of XSB administration. (B) Expression levels of THBD, ARRB1, and FGF1 proteins after 8, 6, and 4 weeks of XSB administration. (C) Schematic representation of the differentially expressed proteins expressed after 4, 6, and 8 weeks of XSB administration to the MI-induced pro- and post- HFrEF mice. All data are expressed as the mean ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: The membranes were blocked with 5 % skim milk powder and incubated with the primary antibodies against huntingtin interacting protein 1 (HIP1, BOSTER, BM5424, 1:1000), arrestin β 1 (ARRB1, ABclonal, A10742, 1:1000), fibroblast growth factor 1 (FGF1, ABclonal, A5900, 1:1000), thrombomodulin (THBD, ABclonal, A4155, 1:500), PTBP1 (BOSTER, A01798, 1:1000), Stromal Interaction Molecule 1 (STIM1, BOSTER, PB9406, 1:1000), GAPDH (Proteintech, 60004–1-Ig, 1:20000) for overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: PTBP1 enhances miR-101-guided AGO2 targeting to MCL1 and promotes miR-101-induced apoptosis

doi: 10.1038/s41419-018-0551-8

Figure Lengend Snippet: PC3 and H1299 cells were transfected with either siControl or one of two individual siRNAs targeting AGO2 (siAGO2#1 and siAGO2#2) for 48 h. a AGO2, PTBP1, MCL1, and β-actin levels were detected by western blotting. Relative MCL1 band intensity was quantified and normalized against β-actin. b Relative MCL1 , PTBP1 , and AGO2 mRNA levels were quantified by RT-qPCR using ACTB as the internal control. Data is shown as mean ± SEM, n = 3. The statistical significance was determined using unpaired Student’s t -test where * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were incubated with anti-AGO2 antibody (RN003M, MBL International, Woburn, MA, USA), anti-PTBP1 antibody (RN011P, MBL International), anti-MCL1 antibody (D35A5, Cell Signaling), and anti-β-actin antibody (PA1-21167, Pierce/Thermo Fisher) at a dilution of 1:1000 at 4 °C overnight.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Control

Journal: Cell Death & Disease

Article Title: PTBP1 enhances miR-101-guided AGO2 targeting to MCL1 and promotes miR-101-induced apoptosis

doi: 10.1038/s41419-018-0551-8

Figure Lengend Snippet: PC3 and H1299 cells were transfected with siControl, siPTBP1, siAGO2, or siAGO2+siPTBP1 for 48 h. a AGO2, PTBP1, MCL1, and β-actin levels were detected by western blotting. Relative MCL1 band intensity was quantified and normalized against β-actin. b Relative MCL1 , AGO2, PTBP1 RNA levels were quantified by RT-qPCR using ACTB as the internal control. For all panels, data is shown as mean ± SEM, n = 3. The statistical significance was determined using unpaired Student’s t -test where n.s. (not significant) p > 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were incubated with anti-AGO2 antibody (RN003M, MBL International, Woburn, MA, USA), anti-PTBP1 antibody (RN011P, MBL International), anti-MCL1 antibody (D35A5, Cell Signaling), and anti-β-actin antibody (PA1-21167, Pierce/Thermo Fisher) at a dilution of 1:1000 at 4 °C overnight.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Control

Journal: Cell Death & Disease

Article Title: PTBP1 enhances miR-101-guided AGO2 targeting to MCL1 and promotes miR-101-induced apoptosis

doi: 10.1038/s41419-018-0551-8

Figure Lengend Snippet: a RIP using IgG (control) or AGO2 antibody was performed in PC3 cells transfected with either siControl or siPTBP1 for 48 h. The amount of MCL1 RNA binding to AGO2 was quantified as percentage of input in IP by RT-qPCR. b Immunoprecipitated AGO2, PTBP1, MCL1, and β-actin protein levels were assessed by western blotting. Data is shown as mean ± SEM, n = 3. The statistical significance was determined using unpaired Student’s t -test where *** p < 0.001.

Article Snippet: Membranes were incubated with anti-AGO2 antibody (RN003M, MBL International, Woburn, MA, USA), anti-PTBP1 antibody (RN011P, MBL International), anti-MCL1 antibody (D35A5, Cell Signaling), and anti-β-actin antibody (PA1-21167, Pierce/Thermo Fisher) at a dilution of 1:1000 at 4 °C overnight.

Techniques: Control, Transfection, RNA Binding Assay, Quantitative RT-PCR, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: PTBP1 enhances miR-101-guided AGO2 targeting to MCL1 and promotes miR-101-induced apoptosis

doi: 10.1038/s41419-018-0551-8

Figure Lengend Snippet: a The CLIP-seq analysis of AGO2 binding events in control or PTBP1 knockdown HELA cells are mapped on MCL1 RNA (GSM1048187 and GSM1048188). Three major peaks with decreased AGO2 binding signals upon PTBP1 knockdown were numbered I, II, and III. An MCL1 mRNA sequence in peak III is highlighted with the miR-101 seed in gray shading. b RIP was performed in PC3 cells transfected with either negative control miRNA (miR-NC) or miR-101 mimic (miR-101) for 24 h. The amount of MCL1 RNA binding to AGO2 (% of input in IP) was quantified by RT-qPCR. d H1299 cells were transfected with either siControl or siPTBP1 for 24 h, and then transfected with either miR-NC or miR-101 for another 24 h. RIP was then performed. The amount of MCL1 RNA binding (% of input in IP) to AGO2 was quantified by RT-qPCR. e The effect of miR-101-induced AGO2 binding was calculated as MCL1 fold enrichment in AGO2-RIP by miR-101 vs. miR-NC. c , f AGO2, PTBP1, MCL1, and β-actin protein levels in RIP were assessed by western blotting. Data is shown as mean ± SEM, n = 3. The statistical significance was determined using an unpaired Student’s t -test where * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were incubated with anti-AGO2 antibody (RN003M, MBL International, Woburn, MA, USA), anti-PTBP1 antibody (RN011P, MBL International), anti-MCL1 antibody (D35A5, Cell Signaling), and anti-β-actin antibody (PA1-21167, Pierce/Thermo Fisher) at a dilution of 1:1000 at 4 °C overnight.

Techniques: Binding Assay, Control, Knockdown, Sequencing, Transfection, Negative Control, RNA Binding Assay, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: PTBP1 enhances miR-101-guided AGO2 targeting to MCL1 and promotes miR-101-induced apoptosis

doi: 10.1038/s41419-018-0551-8

Figure Lengend Snippet: a , b H1299 and PC3 cells were transfected with siControl or siPTBP1 first for 24 h and then transfected with miR-NC or miR-101 for another 24 h. Total cell lysates were collected. PTBP1, MCL1, and β-actin levels were detected by western blotting. Relative MCL1 band intensity was quantified and normalized against β-actin. c , d The effect of miR-101 targeting MCL1 was calculated as the percentage of MCL1 upon miR-101 treatment, compared with miR-NC treatment. Data is shown as mean ± SEM, n = 3. The statistical significance was determined using unpaired Student’s t -test where * p < 0.05; ** p < 0.01.

Article Snippet: Membranes were incubated with anti-AGO2 antibody (RN003M, MBL International, Woburn, MA, USA), anti-PTBP1 antibody (RN011P, MBL International), anti-MCL1 antibody (D35A5, Cell Signaling), and anti-β-actin antibody (PA1-21167, Pierce/Thermo Fisher) at a dilution of 1:1000 at 4 °C overnight.

Techniques: Transfection, Western Blot

Journal: Cell Death & Disease

Article Title: PTBP1 enhances miR-101-guided AGO2 targeting to MCL1 and promotes miR-101-induced apoptosis

doi: 10.1038/s41419-018-0551-8

Figure Lengend Snippet: a A summary diagram of the PTBP1–AGO2/miR-101 axis regulation of MCL1. b In the presence of normal PTBP1 expression, PTBP1 binds to MCL1 and facilitates the association of AGO2/miR-101 with MCL1 mRNA, regulating MCL1 levels. c When PTBP1 expression is suppressed, AGO2/miR-101 interaction with MCL1 is reduced. As a result, MCL1 expression increases.

Article Snippet: Membranes were incubated with anti-AGO2 antibody (RN003M, MBL International, Woburn, MA, USA), anti-PTBP1 antibody (RN011P, MBL International), anti-MCL1 antibody (D35A5, Cell Signaling), and anti-β-actin antibody (PA1-21167, Pierce/Thermo Fisher) at a dilution of 1:1000 at 4 °C overnight.

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: Tissue-specific regulation of gene expression via unproductive splicing

doi: 10.1093/nar/gkad161

Figure Lengend Snippet: Examples of novel tissue-specific USEs. ( A and B ) The results for DCLK2 and IQGAP1 genes, respectively. In each case, boxplots show (left to right) the distribution of ψ, e g , the expression levels of the regulator ( PTBP1 ) and the protein expression level. Statistically significant deviations from the pooled median are marked with asterisks (***FDR <0.001; **FDR <0.01; *FDR <0.05; NS not significant). The color of an asterisk shows whether the deviation is in the expected (green) direction. Tissues are colored by GTEx color codes and sorted in ascending ψ order. The ideograms in each panel show the USE and CLIP peaks of PTBP1 . The subpanels on the right show the results of RT-PCR (top) and RT–qPCR (bottom) experiments. ‘PE+’ and ‘PE–’ denote AS isoforms with and without the poison exon, respectively. The lanes are (left to right) non-treated control (con), treatment with siRNA targeting the firefly luciferase gene (luc), treatment with siRNA targeting PTBP1 , CHX treatment, CHX and luc treatment, and treatment with CHX and siRNA targeting PTBP1 . The code for asterisks is as in the left subpanels.

Article Snippet: Membranes were blocked in Tris-buffered saline (TBS, pH 7.4) containing 5% bovine serum albumin (BSA), washed three times in TBS and incubated with primary anti-PTBP1 antibody (Cloud-Clone Corp., PAC737Hu01), or polyclonal mouse anti-glyceraldehyde phosphate dehydrogenase (GAPDH) [6C5] (Abcam, ab8245) antibodies overnight at 4°C (1:2000 dilution).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Luciferase