Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 1 AChE interacts with RanBPM (A) The self-expression assay of the bait. AH109/pGBKT7-AChEC-terminal cells were cultured on SD/-Trp plate or SD/-Trp/-His plates. (B) Library screening. AH109 clones transfected with pGBKT7-AChEC-terminal and human fetal brain library were cultured on SD/-Ade/-His/-Leu/-Trp/X-a-gal plates. The arrows indicate the positive clones (blue clones). (C) The scheme of the two positive clones (A and C6). The putative SPRY domain (amino acids 212–333) is indicated. (D) Clones A and C6 showed strong lacZ reporter activation. Pos., a clone consisting of BD-p53 and AD-SV40 T-antigen was used as the positive control. Neg., a combination of BD-laminC and AD-SV40 T-antigen was used as the negative control; b-galactosidase activity is expressed as a percentage of the positive control. The data are represented as the mean+ SD of three independent transformations. (E,F) HEK293T cells transfected with the indicated expression plasmid for 24 h; the whole-cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting with anti-HA antibody (top panel). Separate aliquots of the lysates were immunoblotted with anti-HA (middle panel) or anti-Myc (bottom panel) to confirm the expression of HA-RanBPM or Myc-AChEC-terminal (or Myc-AChE), respectively. (G) Kidney tissues from the rat model of ischemia/reperfusion injury were immunoprecipitated using the anti-AChE antibody (E70), and analyzed by immunoblotting with the anti-RanBPM antibody (top panel). Separate aliquots of the lysates were immunoblotted with anti-RanBPM (middle panel) or anti-AChE (E70) (bottom panel) to confirm the expression of RanBPM or AChE, respectively, in the rat kidney tissues.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Expressing, Cell Culture, Library Screening, Clone Assay, Transfection, Activation Assay, Positive Control, Negative Control, Activity Assay, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 3 Subcellular distribution of RanBPM and AChE (A,B) Fractionation of HEK293T cell extracts. HEK293T cells were transfected with the indicated expression plasmids. At 24 h after transfection, the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions and analyzed directly by immunoblotting with the anti-HA (first panel) or anti-Myc antibody (second panel). The purity of the nuclear and cytoplasmic fractions was examined by immunoblotting with anti-histone 3 (fourth panel) and anti-a-tubulin antibody (third panel), respectively. (C) Determination of co-localization of AChE and RanBPM by immunofluorescence. HEK293T cells were transfected with Myc-RanBPM and GFP-AChE. At 24 h after transfection, the cells were fixed and incubated with anti-Myc (red), followed by incubation with Rhodamine-conjugated secondary antibodies. The cells were examined by fluorescence microscopy.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Fractionation, Transfection, Expressing, Western Blot, Incubation, Fluorescence, Microscopy

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 4 Subcellular distribution of RanBPM and AChE after cisplatin treatment (A) HEK293T cell viability was assessed by MTT after treatment with different concentrations of cisplatin for 24 h. The graph shows the mean+ SE of three independent experiments. (B) The transcription level of p73a gene detected by RT–PCR. HEK293T cells were treated with different concentrations of cisplatin for 24 h and then total mRNA was extracted for RT–PCR. (C) Cisplatin of 100 mM or higher induced DNA fragmentation. (D) The amount of RanBPM and AChE in the nuclear fractions increased after treatment with cisplatin. After cotransfection with pCMV-Myc-RanBPM and pEGFP-C1-AChE for 24 h, HEK293T cells were treated with the indicated concentrations of cisplatin for another 24 h. Then, the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions. The cell fractions and the whole-cell lysates were immunoblotted with anti-Myc (left line) or anti-GFP (light line). The graph shows the densitometric values of the proteins in the cytoplasm versus those in the nuclei.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cotransfection

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 5 Nuclear distribution of endogenous RanBPM and AChE after long-term culture (A) Cleaved caspase 3 was detected in long-term cultured HEK293T cells. HEK293T cells were cultured without medium changed for 3 days (3D) or 7 days (7D), and then the whole-cell lysates were immunoblotted with anti-capspase 3, or anti-b-actin antibody. (B) The amount of endogenous RanBPM and AChE in the nuclear fractions was increased after long-term culture. HEK293T cells were cultured without medium changed for 2 days (2D), 5 days (5D) or 8 days (8D), and then the nuclear fractions were immunoblotted with anti-AChE (first panel) or anti-RanBPM antibody (second panel, NB 100–1281). The purity of the nuclear and cytoplasmic fractions was examined by immunoblotting with anti-a-tubulin (third panel) or anti-histone 3 antibody (fourth panel), respectively.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Subcellular Localization of the Aryl Hydrocarbon Receptor Is Modulated by the Immunophilin Homolog Hepatitis B Virus X-associated Protein 2

doi: 10.1074/jbc.m006873200

Figure Lengend Snippet: FIG. 5. Examination of AhR localization and AhR/XAP2 stoichiometry in Hepa-1 cells. A, laser scanning confocal micrographs of AhR in control and 1 nM TCDD-treated Hepa-1 cells visualized with anti-AhR MAb RPT9 and goat anti-mouse IgG conjugated to lissamine-rhodamine sulfonyl chloride, Me2SO (i), 30 min (ii), 1 h (iii), 2 h (iv). B, relative amount of AhR in Hepa-1 cytosolic fraction compared with total AhR (cytosolic 1 nuclear). Cells were treated with 1 nM TCDD for 2 h, harvested, homogenized, and centrifuged to obtain nuclear pellet and crude cytosol (respun to obtain cytoplasm). Nuclear pellet was extracted with MENG (1500 mM NaCl), and soluble extract collected. Aliquots of cytosolic and nuclear extracts were resolved by TSDS-PAGE, blotted to PVDF membrane, and visualized using the RPT1/biotin-goat anti-mouse IgG/125I-labeled streptavidin system. C, sedimentation profile of AhR from cytosolic extract of Hepa-1 cells treated with 1 nM TCDD for 2 h. Extracts were applied to 10–30% sucrose gradient in MENG and fractionated. Fractions were acetone precipitated and analyzed by SDS-PAGE, visualized as in panel B, and bands excised and quantitated in a gamma counter. D, ratios of AhR to XAP2 in Hepa-1 cells in cytosolic extracts (cytosol) and in the immunoprecipitated 9 S AhR (complex).

Article Snippet: The membrane was then analyzed by Western blot with RPT1 (anti-AhR MAb) and anti-ARA9 (anti-XAP2 MAb; Novus Biologicals) primary antibodies, and 125I-labeled goat anti-mouse IgG (PerkinElmer Life Sciences) secondary Ab.

Techniques: Control, Membrane, Labeling, Sedimentation, SDS Page, Immunoprecipitation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation.

doi: 10.1002/advs.202400794

Figure Lengend Snippet: Figure 1. Endogenous AHR and SPHK2 interact in HeLa cells. A,B) HeLa cells treated with or without TCDF were subjected to cytoplasmic/nuclear extract fractionation and immunoprecipitated using AHR antibody and western blotting analysis was performed using an (A) AHR antibody or (B) SPHK2 antibody. C, Control; T, TCDF treated samples. C) After IP of SPHK2, western blotting analysis was performed with AHR, ARNT and SPHK2 antibodies. C, Control; T, TCDF treated samples. D) After IP of ARNT, western blotting analysis was performed with AHR, SPHK2 and ARNT antibodies. C, Control; T, TCDF treated samples. E) Immunofluorescent double staining using anti-AHR and anti-SPHK2 antibodies. The nucleus was counterstained with Hoechst 33 342. Arrows c indicate the cytoplasm, n indicate the nucleus. Bar is 10 μm. Data are representative images from three independent experiments.

Article Snippet: Antibodies: Primary antibodies used were as follows: AHR: AHR Rabbit mAb (D5S6H, Cell Signaling technology), Goat anti-AHR antibody (#NB100-128, Novus Biologicals), Mouse monoclonal Anti-AHR #14- 9854-82, Invitrogen), ARNT: HIF-1b/ARNT Rabbit mAb (D28F3, Cell Signaling Technology), SPHK2: SPHK2 Rabbit mAb (D2V3G, Cell Signaling Technology), Phospho-SPHK2: Anti-Phospho-SphK2 (T614) Antibody (#A01382T614, Bosterbio), SPTLC1: anti-SPTLC1 antibody (ab176706, abcam), SPTLC2: anti-Serine Palmitoyltransferase antibody (ab236900, abcam), SPHK1: SPHK1 (D1H1L) Rabbit mAb (#12 071, Cell Signaling Technology), S1P1: S1P1 Polyclonal Antibody (#PA1-1040, Thermo Scientific), Actinb: β-Actin (13E5) Rabbit mAb (#4970, Cell signaling Technology), GAPDH: GAPDH (D16H11) XP Rabbit mAb (#5174, Cell Signaling Technology), Histon H3: Histone H3 (D1H2) XP Rabbit mAb (#4499, Cell Signaling Technology), HA: HA-Tag Rabbit mAb (C29F4, Cell Signaling Technology).

Techniques: Fractionation, Immunoprecipitation, Western Blot, Control, Double Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation.

doi: 10.1002/advs.202400794

Figure Lengend Snippet: Figure 8. Schematic illustration of the dual function of SPHK2. A) SPHK2 functions as a cofactor for the AHR/ARNT heterodimer on the DRE-containing promoter region of the CYP1A1 gene. B) SPHK2, S1P, and AHR establish a positive feedback mechanism for ceramide de novo biosynthesis metabolism. S1P also enhances AHR recruitment to DREs.

Article Snippet: Antibodies: Primary antibodies used were as follows: AHR: AHR Rabbit mAb (D5S6H, Cell Signaling technology), Goat anti-AHR antibody (#NB100-128, Novus Biologicals), Mouse monoclonal Anti-AHR #14- 9854-82, Invitrogen), ARNT: HIF-1b/ARNT Rabbit mAb (D28F3, Cell Signaling Technology), SPHK2: SPHK2 Rabbit mAb (D2V3G, Cell Signaling Technology), Phospho-SPHK2: Anti-Phospho-SphK2 (T614) Antibody (#A01382T614, Bosterbio), SPTLC1: anti-SPTLC1 antibody (ab176706, abcam), SPTLC2: anti-Serine Palmitoyltransferase antibody (ab236900, abcam), SPHK1: SPHK1 (D1H1L) Rabbit mAb (#12 071, Cell Signaling Technology), S1P1: S1P1 Polyclonal Antibody (#PA1-1040, Thermo Scientific), Actinb: β-Actin (13E5) Rabbit mAb (#4970, Cell signaling Technology), GAPDH: GAPDH (D16H11) XP Rabbit mAb (#5174, Cell Signaling Technology), Histon H3: Histone H3 (D1H2) XP Rabbit mAb (#4499, Cell Signaling Technology), HA: HA-Tag Rabbit mAb (C29F4, Cell Signaling Technology).

Techniques: