Journal: Acta Crystallographica Section F Structural Biology Communications

Article Title: Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail

doi: 10.1107/s2053230x15009243

Figure Lengend Snippet: Figure 1 Crystallization and SDS–PAGE analysis of purified human CRMP-1. (a) Morphology of CRMP-1 crystals. Left, the crystals after crystallization (2 d, 293 K). Right, the crystals after incubation (20 d). (b) Analysis (SDS–PAGE) of CRMP-1 proteins that had not (lane 1) or had (lane 2) been incubated with thrombin protease and a sample taken from a crystallization well containing high-quality CRMP-1 crystals after incubation (20 d at 293 K; lane 3). Samples (3 mg) were characterized using a Tris–glycine gel (12%) and stained with Coomassie Blue. (c) SDS– PAGE analysis of the purified C-terminal tail (residues 472–572) of CRMP-1. Samples were separated with a Tris–tricine (20%) peptide- separation gel and were detected with silver staining.

Article Snippet: Oligomerization analyses were performed with full-length and thrombin-cleaved CRMP-1 (using an analytical Superdex 200 10/300 column from GE Healthcare connected to an ÄKTA purifier system).

Techniques: Crystallization Assay, SDS Page, Incubation, Staining, Silver Staining

Journal: Acta Crystallographica Section F Structural Biology Communications

Article Title: Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail

doi: 10.1107/s2053230x15009243

Figure Lengend Snippet: Figure 2 A representative view of the final 2Fo Fc electron-density map (blue, resolution 3 A˚ , contoured at the 1.0 level), highlighting the fit of residues Arg467–Pro475 to the maps. The nonconsensus amino acid (depicted in red) in this region is Ala473 of human CRMP-1 (phenylalanine in mouse CRMP-1).

Article Snippet: Oligomerization analyses were performed with full-length and thrombin-cleaved CRMP-1 (using an analytical Superdex 200 10/300 column from GE Healthcare connected to an ÄKTA purifier system).

Techniques:

Journal: Acta Crystallographica Section F Structural Biology Communications

Article Title: Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail

doi: 10.1107/s2053230x15009243

Figure Lengend Snippet: Figure 4 Residues that are potentially important in determining interfaces in CRMP. The crystal structures of mouse CRMP-1 (yellow; PDB entry 1kcx; Deo et al., 2004), CRMP-2 (green; PDB entry 2gse; Stenmark et al., 2007), CRMP-4 (cyan; PDB entry 4bkn; Structural Genomics Consor- tium, unpublished work) and CRMP-5 (blue; PDB entry 4b91; Ponnusamy & Lohkamp, 2013) are superimposed onto human CRMP-1 (orange; PDB entry 4b3z). Both interfaces I (a) and II (b) contain conserved and nonconserved residues. The interacting residues are labelled for human CRMP-1; the corresponding residues of mouse CRMP-1, CRMP-2, CRMP-4 and CRMP-5 are given in parentheses.

Article Snippet: Oligomerization analyses were performed with full-length and thrombin-cleaved CRMP-1 (using an analytical Superdex 200 10/300 column from GE Healthcare connected to an ÄKTA purifier system).

Techniques:

Journal: Acta Crystallographica Section F Structural Biology Communications

Article Title: Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail

doi: 10.1107/s2053230x15009243

Figure Lengend Snippet: Figure 6 Oligomerization and characterization of the secondary structure of full- length and thrombin-cleaved CRMP-1. (a) Analytical gel-filtration chromatography of full-length (black line) and thrombin-cleaved (red line) CRMP-1 was performed as described in the Materials and methods; the profiles are superimposed based on the ‘inject’ signal. The elution volumes corresponding to the molecular masses of the protein markers is marked with blue arrows for comparison. Vo and Vt denote the void and total volumes of the column, respectively. Under these operating conditions, the apparent predominant species of full-length and thrombin-cleaved CRMP-1 are tetramers. (b) CD spectra of CRMP-1 (black line) and thrombin-cleaved CRMP-1 (red line) were recorded in the 260–200 nm wavelength region. In the inset in (b), CD spectra of the C-terminal tail of CRMP-1 (amino acids 472–572; grey line), the -helix- rich protein myoglobin (blue line) and the -strand-rich protein concanavalin A (green line) (each protein was dissolved in 10 mM Tris–HCl pH 7.4, 150 mMNaF) are shown in mean residue ellipticity units; the buffer baseline was subtracted.

Article Snippet: Oligomerization analyses were performed with full-length and thrombin-cleaved CRMP-1 (using an analytical Superdex 200 10/300 column from GE Healthcare connected to an ÄKTA purifier system).

Techniques: Chromatography, Comparison, Circular Dichroism, Residue

Journal:

Article Title: COVALENT REGULATION OF ULVWF STRING FORMATION AND ELONGATION ON ENDOTHELIAL CELLS UNDER FLOW CONDITIONS †

doi: 10.1111/j.1538-7836.2008.02991.x

Figure Lengend Snippet: A. ULVWF and plasma VWF multimers were labeled with MPB and immunoprobed with HRP-streptavidin and a polycloncal VWF antibody. Both forms of VWF were labeled with MPB. B. ULVWF and plasma VWF multimers were incubated with the thiol active sepharose 6B beads and the bead captured VWF (containing free thiols) was released by DTT. VWF in supernatant and eluted from the beads was detected by immunoblotting with the polyclonal VWF antibody. The figure is a representative of 6 and 3 separate experiments for plasma VWF and ULVWF, respectively.

Article Snippet: Cell supernatant was collected and the chimera purified through a sepharose 4B gel filtration column (PD-10, Amersham Bioscience, Uppsala, Sweden).

Techniques: Labeling, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene

doi: 10.1016/j.jbc.2021.100601

Figure Lengend Snippet: Downregulation of ADAR1 reduces the expression of P-gp in RPTECs. A , construction of ADAR1-knockdown (KD) RPTECs with stable expression of shRNA. The expression levels of ADAR1-p110 protein were normalized to that of β-ACTIN. Values are the mean with S.D. (n = 3). The value of ADAR1-p110 in mock-transduced RPTECs was set at 1.0. ∗∗ p < 0.01; significant difference between the two groups ( t 4 = 6.988, p = 0.002; unpaired t -test, two-sided). B , the mRNA levels of solute carrier (SLC) transporters in mock-transduced and ADAR1-KD RPTECs. The mRNA levels were assessed by quantitative real-time RT-PCR analysis and their expression levels were normalized to that of 18S rRNA. Values are the mean with S.D. (n = 4). C , the mRNA levels of ABC transporters in mock-transduced and ADAR1-KD RPTECs. The mRNA levels were normalized to that of 18S rRNA. Values are the mean with S.D. (n = 3). ∗∗ p < 0.01, ∗ p < 0.05; significant difference between the two groups ( t 4 = 5.656, p = 0.005 for ABCB1 ; t 4 = 4.175, p = 0.014 for ABCC2 ; unpaired t -test, two-sided). D , The protein levels of P-gp and MRP2 in mock-transduced and ADAR1-KD RPTECs. The protein levels were normalized to that of β-ACTIN. Values are the mean with S.D. (n = 3–4). ∗ p < 0.05; significant difference between the two groups ( t 4 = 2.938, p = 0.043 for P-gp; unpaired t -test, two-sided).

Article Snippet: The membranes were incubated with primary antibodies against ADAR1 (1:1000, sc-73408; Santa Cruz Biotechnology), P-gp (1:1000, C494, Thermo Fisher Scientific), MRP2 (1:1500, sc-5770; Santa Cruz Biotechnology), TBP (1:1000, ab51841; abcam), and β-ACTIN (1:10,000, sc-1616; Santa Cruz Biotechnology).

Techniques: Expressing, Knockdown, shRNA, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene

doi: 10.1016/j.jbc.2021.100601

Figure Lengend Snippet: ADAR1 is involved in circadian regulation of P-gp expression in human RPTECs. A , circadian oscillation of PERIOD2 and BMAL1 mRNA ( left ), and ADAR1 and P-gp protein ( right ) in the kidney of cynomolgus monkeys. The mRNA levels were assessed by quantitative real-time RT-PCR analysis and their expression levels were normalized to that of β-ACTIN . Values are the mean with S.D. (n = 3). The protein levels of β-ACTIN are shown as loading controls. B , temporal expression profiles of PERIOD2 and BMAL1 mRNA in mock-transduced and ADAR1-KD RPTECs after treatment with 100 nM DEX for 2 h. The mRNA levels were assessed by quantitative real-time RT-PCR analysis and their expression levels were normalized to that of 18S rRNA. Values are the mean with S.D. (n = 3). The value of mock-transduced RPTECs before DEX treatment (Pre) was set at 1.0. There were significant time-dependent variations in PERIOD2 and BMAL1 mRNA expression ( F 13,28 = 56.917, p < 0.001 and F 13,28 = 46.243, p < 0.001 for PERIOD2 in mock-transduced and ADAR1-KD RPTECs, respectively; F 13,28 = 14.565, p < 0.001 and F 13,28 = 5.157, p < 0.001 for BMAL1 in mock-transduced and ADAR1-KD RPTECs, respectively; ANOVA). ∗∗ p < 0.01; significant difference between the two groups ( t 4 = 38.454, p < 0.001; unpaired t -test, two-sided). C , temporal protein expression profiles of ADAR1 in mock-transduced and ADAR1-KD RPTECs after treatment with 100 nM DEX. The protein levels were normalized to that of β-ACTIN. Values are the mean with S.D. (n = 4). The value of mock-transduced RPTECs before DEX treatment (Pre) was set at 1.0. There were significant time-dependent variations ( F 5,18 = 10.414, p < 0.001 for mock-transduced; F 5,18 = 8.214, p < 0.001 for ADAR1-KD; ANOVA). ∗∗ p < 0.01; significant difference between the two groups ( t 6 = 4.641, p = 0.004; unpaired t -test, two-sided). D , temporal expression profiles of P-gp in mock-transduced and ADAR1-KD RPTECs after treatment with 100 nM DEX. The protein levels were normalized to that of β-ACTIN. Values are the mean with S.D. (n = 4). The value of mock-transduced RPTECs before DEX treatment (Pre) was set at 1.0. There was a significant time-dependent variation in mock-transduced RPTECs ( F 5,18 = 2.899, p = 0.043 for mock-transduced; ANOVA). ∗ p < 0.05; significant difference between the two groups ( t 6 = 2.617, p = 0.040; unpaired t -test, two-sided). In panel C and D , β-ACTIN was reused as loading control because the same protein samples were used for Western blotting for detection of ADAR1 and P-gp. E , intracellular accumulation of digoxin in mock-transduced and ADAR1-KD RPTECs 32 and 44 h after DEX treatment. Concentrations of digoxin were measured by LC-MS/MS analysis 1 h after incubation with the drug. Values are the mean with S.D. (n = 4). ∗ p < 0.05; significant difference between the two groups ( F 3,12 = 7.487, p = 0.004; ANOVA with Tukey–Kramer’s post hoc test).

Article Snippet: The membranes were incubated with primary antibodies against ADAR1 (1:1000, sc-73408; Santa Cruz Biotechnology), P-gp (1:1000, C494, Thermo Fisher Scientific), MRP2 (1:1500, sc-5770; Santa Cruz Biotechnology), TBP (1:1000, ab51841; abcam), and β-ACTIN (1:10,000, sc-1616; Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: The Journal of Biological Chemistry

Article Title: RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene

doi: 10.1016/j.jbc.2021.100601

Figure Lengend Snippet: ADAR1 regulates the time-dependent differences in the alternative splicing of the human ABCB1 gene. A , schematic diagram of the retention of intron 27 of the ABCB1 gene. Intron-retaining ABCB1 mRNA may be degraded through nonsense-mediated mRNA decay (NMD). B , ADAR1 is involved in the time-dependent differences in the production of retained-intron 27 ABCB1 transcripts. Cells were transfected with the ex27–ex28 minigene and treated with DEX. Minigene-derived normal splicing transcripts and retained-intron transcripts were assessed by semiquantitative RT-PCR at the indicated time points after DEX treatment. The percentage of retained intron was calculated by dividing the signal intensity of retained intron by the summed signal intensity of retained intron and normal splicing transcripts. Values are the mean with S.D. (n = 3). ∗ p < 0.05; significant difference between the two groups ( F 3,8 = 11.373, p = 0.003; ANOVA with Tukey–Kramer’s post hoc test). C , ADAR1 binds to intron 27 of ABCB1 pre-mRNA. RPTECs were transfected with the ABCB1 ex27–ex28 minigene, and then RIP assay was conducted at 24 h posttransfection. Upper panel shows western blotting analysis of ADAR1-RIP immunoprecipitates. Lower panel shows quantitative real-time RT-PCR analysis of ADAR1-RIP immunoprecipitates using intron 27 specific primers listed in Table 1 . Values are the mean with S.D. (n = 3). ∗∗ p < 0.01; significant difference between the two groups ( t 4 = 12.964, unpaired t -test, two-sided). D , retained-intron 27 transcripts of the ABCB1 gene are degraded by NMD. Cells were transfected with the ex27–ex28 minigene and treated with 1 μM NMDI-14 for 6 h. Minigene-derived normal splicing transcripts and retained-intron transcripts were assessed by semiquantitative RT-PCR. Values are the mean with S.D. (n = 4). ∗∗ p < 0.01; significant difference between the two groups ( t 6 = 8.022, unpaired t -test, two-sided). E , the stability of retained-intron 27 transcript of the ABCB1 gene. Cells were transfected with the ex27–ex28 minigene. Minigene-derived normal splicing transcripts and retained-intron transcripts were assessed by semiquantitative RT-PCR. The mRNA levels were normalized to that of 18S rRNA. Values are the mean with S.D. (n = 3). The value at 0 h (the time of the initiation of ActD) was set at 1.0. ∗ p < 0.05; significant difference between the two groups at the corresponding time point ( F 5,12 = 13.641, p < 0.001; ANOVA with Tukey–Kramer’s post hoc test). F and G , NMD inhibition restores levels of ABCB1 mRNA and P-gp in ADAR1-KD RPTECs. Mock-transduced and ADAR1-KD cells were treated with 1 μM NMDI-14 for 24 h. The mRNA levels were assessed by quantitative real-time RT-PCR, and their levels were normalized to that of 18S rRNA. The levels of P-gp were normalized that of β-ACTIN. Values are the mean with S.D. (n = 4). The value of vehicle (0.01% DMSO)-treated mock cells was set at 1.0. ∗∗ p < 0.01, ∗ p < 0.05; significant difference between the indicated groups ( F 3,12 = 15.727, p < 0.001 for ABCB1 mRNA; F 3,12 = 37.583, p < 0.001 for P-gp; ANOVA with Tukey–Kramer’s post hoc test). H , intracellular accumulation of digoxin in NMDI-14-treated ADAR1-KD RPTECs. Concentrations of digoxin were measured by LC-MS/MS analysis 1 h after incubation with the drug. Values are the mean with S.D. (n = 6). ∗∗ p < 0.01; significant difference between the two groups ( t 10 = 4.868, unpaired t -test, two-sided).

Article Snippet: The membranes were incubated with primary antibodies against ADAR1 (1:1000, sc-73408; Santa Cruz Biotechnology), P-gp (1:1000, C494, Thermo Fisher Scientific), MRP2 (1:1500, sc-5770; Santa Cruz Biotechnology), TBP (1:1000, ab51841; abcam), and β-ACTIN (1:10,000, sc-1616; Santa Cruz Biotechnology).

Techniques: Alternative Splicing, Transfection, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Inhibition, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: The Journal of Biological Chemistry

Article Title: RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene

doi: 10.1016/j.jbc.2021.100601

Figure Lengend Snippet: Primer sets for quantitative and semiquantitative RT-PCR analysis of gene expression

Article Snippet: The membranes were incubated with primary antibodies against ADAR1 (1:1000, sc-73408; Santa Cruz Biotechnology), P-gp (1:1000, C494, Thermo Fisher Scientific), MRP2 (1:1500, sc-5770; Santa Cruz Biotechnology), TBP (1:1000, ab51841; abcam), and β-ACTIN (1:10,000, sc-1616; Santa Cruz Biotechnology).

Techniques: