Journal: Theranostics

Article Title: PIM1 instigates endothelial-to-mesenchymal transition to aggravate atherosclerosis.

doi: 10.7150/thno.102597

Figure Lengend Snippet: Figure 8. NDRG1 and PTBP1 collaborate to promote EndMT. (A) The Nuclear and cytoplasm proteins of input, IgG and anti-His-Tag were purified and size fractionated on 10% SDS-PAGE. The gel was stained by coomassie brilliant blue staining. (B) The content of NDRG1 was analyzed by NDRG1 antibody. (C) The Co-IP experiment detecting the interaction between NDRG1 and PTBP1 in nucleus from HUVEC treated with H2O2 (200 μM) and TGF-β (50 ng/mL, 48 h). (D, E) Molecular simulations and protein docking of NDRG1 and PTBP1. (F) Schematic diagrams of 6*His-Tagged full-length (WT) NDRG1, and their various deletion mutants (180-294aa, and 326-394aa) (Top). HEK 293T cells were co-transfected with His-Tagged NDRG1 or its deletion mutants or vectors, and whole cell lysates were assessed by immunoprecipitation followed by immunoblotting with

Article Snippet: PIM1 protein (HY-P701745, MCE), PTBP1 protein (Ag28404, Proteintech), and small molecule Max-40279 (HY-145723, MCE; 500 nM) binding activities were generated with the SPR system, and the binding signal was exhibited by the response (RU) value.

Techniques: Purification, SDS Page, Staining, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Multilevel integrative transcriptome analyses in humans and humanized mice define in vivo human lncRNA metabolic regulators

doi: 10.1101/2020.01.01.884023

Figure Lengend Snippet: (A) RNA ploy II ChIP analyses in liver tissues of humanized mice receiving adenovirus for control (sh-lacZ, n=3) or knocking down of hLMR1 (sh-hLMR1, n=3). (B) Left: western blot analysis of PTBP1 in hLMR1 pulldown, right: expression of hLMR1 in PTBP1 RIP (RNA immunoprecipitation) in humanized liver. (C) HMGCS1 promoter-driven luciferase reporter assay in 293A cells (n=3 for each group). Data are representative results of three independent experiments. (D) PTBP1 ChIP analyses in liver tissues of humanized mice receiving adenovirus for control (sh-lacZ, n=3) or knocking down of hLMR1 (sh-hLMR1, n=3). Error bars represent SEM, * p<0.05.

Article Snippet: Full-length PTBP1 expression vector and control vector were purchased from OriGene (Cat: RC201779 and PS100001).

Techniques: Western Blot, Expressing, Immunoprecipitation, Luciferase, Reporter Assay

Journal: bioRxiv

Article Title: Multilevel integrative transcriptome analyses in humans and humanized mice define in vivo human lncRNA metabolic regulators

doi: 10.1101/2020.01.01.884023

Figure Lengend Snippet: (A) Gene expression in the liver of regular mice receiving lacZ shRNA (sh-lacZ, n=9), or shRNA for Ptbp1 (sh-Ptbp1, n=7). (B) Gene expression in the liver of regular mice receiving adenovirus for control (Ad-Vector, n=5) or expression of hLMR1 (Ad-hLMR1, n=6). (C) Plasma (left) and liver (right) cholesterol levels in regular mice receiving adenovirus for control (Ad-Vector, n=9) or expressing of hLMR1 (Ad-hLMR1, n=9). Error bars represent SEM, * p<0.05.

Article Snippet: Full-length PTBP1 expression vector and control vector were purchased from OriGene (Cat: RC201779 and PS100001).

Techniques: Expressing, shRNA, Plasmid Preparation

Journal: BMC genomics

Article Title: PAIP1 binds to pre-mRNA and regulates alternative splicing of cancer pathway genes including VEGFA.

doi: 10.1186/s12864-024-10530-9

Figure Lengend Snippet: Fig. 5 Protein interaction of PAIP1 and splicing factors. (A) PAIP1 interactions with proteins are identified. Functional analysis of PAIP1 interacting proteins, the top 10 GO biological process terms and KEGG pathway are shown. (B) Network diagram of the RNA splicing proteins identified by coIP MS/MS with PAIP1 antibody. The protein-protein interaction network analyzed by STRING (https://string-db.org/) and then visualized using the Cytoscape program (http://cytoscape.org/). Circle denote those interaction proteins are spliceosome, square denote those interaction proteins are alternative polyadenyl ation, hexagon denote those interaction proteins are splicing factors, rhombus denote those interaction protein is exon junction complex. (C) WB and quantitative results for PTBP1 and HNRNPL in shPAIP1 and NC samples. NC: negative control; Sh: PAIP1 knockdown; ns: non-significant

Article Snippet: The PTBP1 and HNRNPL proteins were detected using primary antibodies against PTBP1 (12582-1-AP, Proteintech, 1:1,000) and HNRNPL (18354-1-AP, Proteintech, 1:1000), respectively.

Techniques: Functional Assay, Tandem Mass Spectroscopy, Negative Control, Knockdown

Journal: Nucleic Acids Research

Article Title: Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells

doi: 10.1093/nar/gkag112

Figure Lengend Snippet: gRNA-dependent off-target binding alters the expression of essential genes and induces substantial alterations in cell proliferation. ( A – D ) RT-qPCR and western blot analyses of on-target EZH2 and three gRNA-dependent off-target genes bound by the PspCas13b-EZH2-g2 complex, with comparisons between non-targeting and EZH2-g2 targeting conditions. ( E – H ) RT-qPCR and western blot analyses of on-target EZH2 and three gRNA-dependent off-target genes bound by the RfxCas13d-EZH2-g2 complex, with comparisons between non-targeting and EZH2-g2 targeting conditions. RT-qPCR data are presented as mean ± SD ( n = 3). Relative changes in protein abundance were quantified by densitometric analysis and are indicated as red numbers at the bottom of each panel. ( I ) Proliferation of cells expressing PspCas13b (left panel) or RfxCas13d (right panel) with either targeting gRNAs or a non-targeting control gRNA (gNT), measured by CCK-8 assay. Proliferation rates were measured by absorbance at days 1–5 and normalized to that at day 1. Data are presented as mean ± SD ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001. ns, not significant. ( J ) Apoptosis analysis of cells expressing PspCas13b (left panel) or RfxCas13d (right panel) with either targeting gRNAs or non-targeting control gRNA (gNT). Data are presented as mean ± SD ( n = 3). ns, not significant.

Article Snippet: The following antibodies were used for western blot analysis in this study: PTBP1 polyclonal antibody (Proteintech, 12582-1-AP), RHOA polyclonal antibody (Proteintech, 10749-1-AP), CTSH polyclonal antibody (Proteintech, 10315-1-AP), CDK19 polyclonal antibody (Proteintech, 13761-1-AP), HMGN2 polyclonal antibody (Proteintech, 10953-1-AP), ACVR2B antibody (Abmart, T58048 ), NUCKS1 polyclonal antibody (Proteintech, 12023-2-AP), HMGA1 polyclonal antibody (Proteintech, 29895-1-AP), EZH2 polyclonal antibody (Proteintech, 21800-1-AP), EFNA3 polyclonal antibody (Proteintech, 12480-1-AP), HPSE polyclonal antibody (Proteintech, 24529-1-AP), SPG7 polyclonal antibody (Proteintech, 27801-1-AP), TRAF3 polyclonal antibody (Proteintech, 18099-1-AP), MDK polyclonal antibody (Proteintech, 11009-1-AP), SCRN1 polyclonal antibody (Proteintech, 14303-1-AP), HA-Tag (26D11) mAb (Abmart, M20003L), GAPDH (3B3) mAb (Abmart, M20006L), ACSL1 polyclonal antibody (Proteintech, 13989-1-AP), FASN polyclonal antibody (Proteintech, 10624-2-AP), RPL14 polyclonal antibody (Proteintech, 14991-1-AP), ACOX1 polyclonal antibody (Proteintech, 10957-1-AP), ACO2 polyclonal antibody (Proteintech, 11134-1-AP), EIF2D polyclonal antibody (Proteintech, 12840-1-AP).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Control, CCK-8 Assay