Journal:

Article Title: Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

doi: 10.1038/sj.emboj.7600275

Figure Lengend Snippet: 7SK snRNA interacts with HEXIM1 in yeast three-hybrid assays. β-Galactosidase activities in yeast three-hybrid (A, C, D), modified three-hybrid (B) and two-hybrid (D) assays. Proteins fused to the Gal4 activation or LexA DNA-binding domains and RNA fused to the MS2-binding sites are indicated in the left panels. Pools of more than 10 transformed yeast colonies grown on selective medium were assayed for β-galactosidase activities and quantified in arbitrary units (a.u.). Interaction between HIV TAR RNA and Tat was used as a positive control. (A) Three-hybrid assay testing truncated 7SK RNAs for interaction with full-length CDK9, cyclin T1 or HEXIM1. (B) Modified three-hybrid testing the capacity of HEXIM1 to link 7SK RNA to CDK9 or cyclin T1. The additional genuine protein is indicated in the central column. (C) Three-hybrid assay testing 7SK(1–175) for interaction with HEXIM1 deletion mutants. (D) Three-hybrid and two-hybrid assays testing 7SK(1–175) and cyclin T1 respectively for interaction with HEXIM1(ILAA).

Article Snippet: Antibodies Antibodies used were rabbit anti-cyclin T1 (H-245, Santa Cruz), goat anti-cyclin T1 (T-18, Santa Cruz), anti-CDK9 mouse monoclonal (D-7, Santa Cruz), rabbit anti-CDK9 (C-20, Santa Cruz), rabbit anti-HEXIM1 (C4) ( Michels et al , 2003 ), anti-Flag mouse monoclonal (M2, Sigma) and anti-GST HRP-conjugated protein (Sigma).

Techniques: Modification, Activation Assay, Binding Assay, Transformation Assay, Positive Control, Hybrid Assay

Journal:

Article Title: Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

doi: 10.1038/sj.emboj.7600275

Figure Lengend Snippet: In vitro binding of HEXIM1 to P-TEFb requires 7SK snRNA. Proteins were detected by Western blot with anti-CDK9, anti-cyclin T1 or anti-HEXIM1 antibodies. (A) Left: Glutathione beads coated with GST-HEXIM1(1–359) were incubated with cell extracts in the presence of increasing concentrations of 7SK (0–80 nM) or U2 RNA (0–160 nM). Right: RNase A was added (+) or not (−) to HEXIM1/7SK/P-TEFb complexes preformed on the beads. Inputs (I), supernatants (S) and beads (B) were probed for cyclin T1 and CDK9. (B) An extract of actinomycin-treated HeLa cells was used to immunoprecipitate P-TEFb with anti-cyclin T1, which was incubated with purified His10-HEXIM1 and increasing concentrations of 7SK (nM) or U2 (nM) RNA.

Article Snippet: Antibodies Antibodies used were rabbit anti-cyclin T1 (H-245, Santa Cruz), goat anti-cyclin T1 (T-18, Santa Cruz), anti-CDK9 mouse monoclonal (D-7, Santa Cruz), rabbit anti-CDK9 (C-20, Santa Cruz), rabbit anti-HEXIM1 (C4) ( Michels et al , 2003 ), anti-Flag mouse monoclonal (M2, Sigma) and anti-GST HRP-conjugated protein (Sigma).

Techniques: In Vitro, Binding Assay, Western Blot, Incubation, Purification

Journal:

Article Title: Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

doi: 10.1038/sj.emboj.7600275

Figure Lengend Snippet: The KHRR motif is involved in in vitro and in vivo formation of the P-TEFb/HEXIM1/7SK RNA complex. (A) Pull-down assay of GST, GST-HEXIM1 WT (WT) and GST-HEXIM1(ILAA) with (+) or without (−) addition of 7SK RNA (80 nM). GST (fusion) proteins bound to glutathione beads were probed with anti-GST. (B) HeLa cells transiently transfected with Flag-HEXIM1(WT), Flag-HEXIM1(ILAA), Flag-HEXIM1(150–359) or Flag-HEXIM1(156–359) were processed for immunofluorescence with anti-Flag antibodies. Nuclei were stained with DAPI. (C) HeLa cells transfected with Flag-HEXIM1(WT), Flag-HEXIM1(ILAA), Flag-HEXIM1(150–359) or Flag-HEXIM1(156–359) or an empty vector (control) were treated (+) or not (−) with actinomycin D (ActD), lysed and immunoprecipitated with anti-Flag antibodies. Proteins in the extracts (inputs) or immunoprecipitated (beads) were probed with anti-Flag, anti-cyclin T1 and anti-CDK9 antibodies. 7SK RNA was detected by Northern blot.

Article Snippet: Antibodies Antibodies used were rabbit anti-cyclin T1 (H-245, Santa Cruz), goat anti-cyclin T1 (T-18, Santa Cruz), anti-CDK9 mouse monoclonal (D-7, Santa Cruz), rabbit anti-CDK9 (C-20, Santa Cruz), rabbit anti-HEXIM1 (C4) ( Michels et al , 2003 ), anti-Flag mouse monoclonal (M2, Sigma) and anti-GST HRP-conjugated protein (Sigma).

Techniques: In Vitro, In Vivo, Pull Down Assay, Transfection, Immunofluorescence, Staining, Plasmid Preparation, Immunoprecipitation, Northern Blot

Journal:

Article Title: Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

doi: 10.1038/sj.emboj.7600275

Figure Lengend Snippet: Involvement of the PYNT motif in HEXIM1 C-terminal domain in in vivo and in vitro binding to P-TEFb. (A) Glutathione beads coated with GST-HEXIM1 full-length (WT) or truncated proteins were incubated with cell extracts with (+) or without (−) 80 nM 7SK RNA. GST-HEXIM1 proteins bound to the beads were detected by Coomassie blue staining. (B) Full-length (1–359) or truncated (181–359) GST-HEXIM proteins with or without the PYND or PDND mutation were tested for binding to P-TEFb in the presence (+) and absence (−) of 80 nM 7SK. (C) Cells transfected with empty vector (control) or Flag-HEXIM1 with or without the PYND or PDND mutations were treated (+) or not (−) with actinomycin D (ActD), lysed and immunoprecipitated with anti-Flag. Beads were probed for cyclin T1, CDK9 and 7SK. Extracts were probed with anti-Flag (inputs).

Article Snippet: Antibodies Antibodies used were rabbit anti-cyclin T1 (H-245, Santa Cruz), goat anti-cyclin T1 (T-18, Santa Cruz), anti-CDK9 mouse monoclonal (D-7, Santa Cruz), rabbit anti-CDK9 (C-20, Santa Cruz), rabbit anti-HEXIM1 (C4) ( Michels et al , 2003 ), anti-Flag mouse monoclonal (M2, Sigma) and anti-GST HRP-conjugated protein (Sigma).

Techniques: In Vivo, In Vitro, Binding Assay, Incubation, Staining, Mutagenesis, Transfection, Plasmid Preparation, Immunoprecipitation

Journal:

Article Title: Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

doi: 10.1038/sj.emboj.7600275

Figure Lengend Snippet: Binding of P-TEFb to GST-HEXIM1 represses its kinase activity. P-TEFb from a cell extract was retained on glutathione beads coated with WT (WT) or truncated (181–359) GST-HEXIM1 with (−) or without (+) 80 nM 7SK. As a control, P-TEFb was immunoprecipitated from a cell extract with anti-cyclin T1 (IP). (A) P-TEFb retained on beads was analyzed using anti-cyclin T1 and CDK9 antibodies. For an accurate comparison, increasing amounts of material (μl) were loaded on the gels. (B) The reactions were analyzed by SDS–PAGE using autoradiography. (C) 32P incorporation into the CTD4 peptide was quantified in arbitrary units (a.u.) and plotted versus time (min). This experiment was performed three times with similar results. A typical experiment is shown.

Article Snippet: Antibodies Antibodies used were rabbit anti-cyclin T1 (H-245, Santa Cruz), goat anti-cyclin T1 (T-18, Santa Cruz), anti-CDK9 mouse monoclonal (D-7, Santa Cruz), rabbit anti-CDK9 (C-20, Santa Cruz), rabbit anti-HEXIM1 (C4) ( Michels et al , 2003 ), anti-Flag mouse monoclonal (M2, Sigma) and anti-GST HRP-conjugated protein (Sigma).

Techniques: Binding Assay, Activity Assay, Immunoprecipitation, SDS Page, Autoradiography

Journal:

Article Title: Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

doi: 10.1038/sj.emboj.7600275

Figure Lengend Snippet: Binding of HEXIM1 to P-TEFb represses its kinase activity. P-TEFb immunoprecipitated with anti-cyclin T1 was incubated with buffer (control), or recombinant histidine-tagged HEXIM1, or T7-transcribed 7SK RNA (80 nM), or a combination of both for 1 h at 21°C. Kinase activity was assayed on the beads as in Figure 5. When indicated, RNase A was added to the kinase assay. This experiment was performed twice with similar results. A typical experiment is shown.

Article Snippet: Antibodies Antibodies used were rabbit anti-cyclin T1 (H-245, Santa Cruz), goat anti-cyclin T1 (T-18, Santa Cruz), anti-CDK9 mouse monoclonal (D-7, Santa Cruz), rabbit anti-CDK9 (C-20, Santa Cruz), rabbit anti-HEXIM1 (C4) ( Michels et al , 2003 ), anti-Flag mouse monoclonal (M2, Sigma) and anti-GST HRP-conjugated protein (Sigma).

Techniques: Binding Assay, Activity Assay, Immunoprecipitation, Incubation, Recombinant, Kinase Assay

Journal:

Article Title: Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

doi: 10.1038/sj.emboj.7600275

Figure Lengend Snippet: Analysis of P-TEFb inhibition by 7SK and HEXIM1 in a defined system. Kinase assays were performed with recombinant P-TEFb and HEXIM1 proteins, and T7-transcribed 7SK RNA. The reactions were analyzed by SDS–PAGE using autoradiography (inset) and quantified. (A) Using DSIF as substrate, the kinase reactions contained increasing amounts of a mixture of HEXIM1 and 7SK (8:1 molar ratio) and P-TEFb (containing cyclin T1 or T2a). (B) Using RNAPII as substrate, kinase assays were performed with cyclin T2a-containing P-TEFb and the indicated amounts of HEXIM1 and/or 7SK. When HEXIM1 and 7SK are both present, they are in an equal molar ratio with the indicated amounts of each individually. (C) Using RNAPII as substrate, kinase assays with cyclin T2a-containing P-TEFb and the indicated HEXIM1 proteins and/or 7SK were performed. HEXIM1 WT and mutant proteins (and 7SK) were present at either 5 or 2 pmol per reaction.

Article Snippet: Antibodies Antibodies used were rabbit anti-cyclin T1 (H-245, Santa Cruz), goat anti-cyclin T1 (T-18, Santa Cruz), anti-CDK9 mouse monoclonal (D-7, Santa Cruz), rabbit anti-CDK9 (C-20, Santa Cruz), rabbit anti-HEXIM1 (C4) ( Michels et al , 2003 ), anti-Flag mouse monoclonal (M2, Sigma) and anti-GST HRP-conjugated protein (Sigma).

Techniques: Inhibition, Recombinant, SDS Page, Autoradiography, Mutagenesis

Journal: Scientific reports

Article Title: 9-oxo-ODAs suppresses the proliferation of human cervical cancer cells through the inhibition of CDKs and HPV oncoproteins.

doi: 10.1038/s41598-023-44365-3

Figure Lengend Snippet: Figure 2. Results of transcriptome and pathway analyses of HeLa cells treated or untreated with 9-oxo- ODAs. (a) PCA plots for RNA sequencing. (b) Volano plots for RNA sequencing. (c) Heat map of genes with significantly altered expression levels. (d) DAVID KEGG pathway analysis list associated with downregulated genes for 9-oxo-ODAs vs control. (e) List of genes related to cell cycle pathways. The list includes cyclin- dependent kinase 1 (CDK1). (f) DAVID KEGG pathway analysis list of associated upregulated genes for 9-oxo- ODAs vs control. (g) Results of GSEA enrichment analysis and representative pathways.

Article Snippet: The following antibodies (Abs) were utilized: anti-CDK1 polyclonal antibody (catalog 19532–1-AP, Proteintech, Rosemont, USA), anti-CDK2 monoclonal antibody (catalog ab32147, Abcam, Cambridge, UK), and anti-GAPDH monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA).

Techniques: RNA Sequencing, Expressing, Control

Journal: Scientific reports

Article Title: 9-oxo-ODAs suppresses the proliferation of human cervical cancer cells through the inhibition of CDKs and HPV oncoproteins.

doi: 10.1038/s41598-023-44365-3

Figure Lengend Snippet: Figure 4. Effects of 9-oxo-ODA treatment on CDK1 and CDK2 expression and cell cycle. (a, b) Relative expression levels of (a) CDK1 and (b) CDK2 mRNA in HeLa and SiHa cells treated with 9-oxo-ODAs determined using qPCR. (c, d) Immunoblot analysis of (c) CDK1 and (d) CDK2 proteins in HeLa cells treated with 9-oxo-ODAs. These blots were cut prior to hybridization with antibodies, and the blots are presented in Supplementary Fig. 2. (e,f) Effects of suppressing (e) CDK1 and (f) CDK2 mRNA expression on HeLa cell proliferative capacity. (g) Effects of 9-oxo-ODA treatment on cell cycle in HeLa and SiHa cells determined using flow cytometry.

Article Snippet: The following antibodies (Abs) were utilized: anti-CDK1 polyclonal antibody (catalog 19532–1-AP, Proteintech, Rosemont, USA), anti-CDK2 monoclonal antibody (catalog ab32147, Abcam, Cambridge, UK), and anti-GAPDH monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Western Blot, Hybridization, Flow Cytometry

Journal: Scientific reports

Article Title: 9-oxo-ODAs suppresses the proliferation of human cervical cancer cells through the inhibition of CDKs and HPV oncoproteins.

doi: 10.1038/s41598-023-44365-3

Figure Lengend Snippet: Figure 5. Effects of 9-oxo-ODA treatment on HPV oncoprotein expression and downstream CDK1 and CDK2 expression. 9-oxo-ODAs suppress HPV oncoproteins, including (a) HPV16-E6, (b) HPV16-E7, (c) HPV18-E6, and (d) HPV18-E7 in a concentration-dependent manner. (e) siRNA-mediated knockdown of E6 reduces mRNA expression of E6, E7, CDK1, and CDK2. (f) siRNA-mediated knockdown of CDK1 does not affect mRNA expression of E6 and E7. (g) siRNA-mediated knockdown of CDK2 does not affect mRNA expression of E6 and E7.

Article Snippet: The following antibodies (Abs) were utilized: anti-CDK1 polyclonal antibody (catalog 19532–1-AP, Proteintech, Rosemont, USA), anti-CDK2 monoclonal antibody (catalog ab32147, Abcam, Cambridge, UK), and anti-GAPDH monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Concentration Assay, Knockdown

Journal: Scientific reports

Article Title: 9-oxo-ODAs suppresses the proliferation of human cervical cancer cells through the inhibition of CDKs and HPV oncoproteins.

doi: 10.1038/s41598-023-44365-3

Figure Lengend Snippet: Figure 6. Antitumor effects of 9-oxo-ODAs on HeLa cells ex vivo and in vivo. (a) Schematic diagram of tissue collection from cervical cancer clinical specimens and ex vivo assay. (b) Representative immunohistochemistry images of CDK1, CDK2, and cleaved-caspase 3 in cervical cancer specimens treated with 9-oxo-ODAs or CDDP. (c-e) Positive rates of immunohistochemical staining for (c) CDK1, (d) cleaved-caspase 3, and (e) CDK2 (n = 9). Cutoff values were set for each and evaluated using QuPath. Due to missing data, n = 6 for CDDP 50 μM. (f) For in vivo analysis, HeLa-ip4 cells were inoculated into the pelvic cavity of mice followed by treatment with 9-oxo-ODAs or vehicle. (g) Comparison of tumor length diameter in 9-oxo-ODAs and vehicle groups.

Article Snippet: The following antibodies (Abs) were utilized: anti-CDK1 polyclonal antibody (catalog 19532–1-AP, Proteintech, Rosemont, USA), anti-CDK2 monoclonal antibody (catalog ab32147, Abcam, Cambridge, UK), and anti-GAPDH monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Ex Vivo, In Vivo, Immunohistochemistry, Immunohistochemical staining, Staining, Comparison

Journal: Journal of neurochemistry

Article Title: Microarray analysis of selected genes in neural stem and progenitor cells.

doi: 10.1046/j.1471-4159.2002.01260.x

Figure Lengend Snippet: Fig. 2 RT–PCR analysis for rat E10.5 neural tubes. Total RNA harvested from rat E10.5 neural tubes was reverse transcribed and a subset of genes detected by array hybridization was tested by RT-PCR. (a–c) A subset of the results. Expression was consistent between the two independent methods of analysis except for EGFR expression. Expression of EGFR was seen by array analysis but was not detected by RT–PCR of E10.5 neural tubes (d). How- ever, it was seen at E14.5. Immunocyto- chemistry using an EGFR antibody (e; immunocytochemistry) showed expres- sion by differentiated cells but not by NSCs; see ventricular zone in (e). 1, E10.5 neural tubles; 2, E14.5 neural tubes. Bar, 400 lm.

Article Snippet: Polyclonal antibodies against a-catenin and epidermal growth factor receptor (EGFR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Hybridization, Expressing, Immunocytochemistry

Journal: Oncogene

Article Title: Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells.

doi: 10.1038/sj.onc.1205918

Figure Lengend Snippet: Figure 3 (a) M1 cells were treated with 50 mM PD98059 and sti- mulated with LIF for 1 h. Cell lysates were immunoblotted with anti-phospho-MAPK antibody (apMAPK) (Cell Signaling) or anti-MAPK antibody (aMAPK) (Santa Cruz Biotechnology). (b) M1 cells were stimulated with LIF plus 50 mM PD98059 or (c) LIF plus 10 ng/ml cyclohexamide (CHX) and induction of BATF mRNA was examined by RNA blot hybridization

Article Snippet: Interestingly, in parallel with the expression pattern of BATF, which peaks between 3 and 6 h after LIF exposure (Figure Figure 2 (a) M1 cells, M1/705F cells and M1/JAB cells were stimulated with LIF for 1 h and cell lysates were immunoblotted with anti-phospho-Y705 Stat3 antibody (ap705YStat3) (New England Biolabs) or anti-Stat3 antibody (aStat3) (New England BioLabs). (b) M1 cells, M1/705F cells and M1/JAB cells were stimulated with LIF for 1 h and induction of BATF mRNA was analysed by RNA blot hybridization. (c) M1/Stat3ER cells were stimulated with 4HT for 3 h and induction of BATF mRNA was analysed by RNA blot hybridization Figure 3 (a) M1 cells were treated with 50 mM PD98059 and stimulated with LIF for 1 h. Cell lysates were immunoblotted with anti-phospho-MAPK antibody (apMAPK) (Cell Signaling) or anti-MAPK antibody (aMAPK) (Santa Cruz Biotechnology). (b) M1 cells were stimulated with LIF plus 50 mM PD98059 or (c) LIF plus 10 ng/ml cyclohexamide (CHX) and induction of BATF mRNA was examined by RNA blot hybridization Oncogene (legend overleaf) Oncogene 1f), there is a transient and reproducible decrease in AP-1 luciferase activity.

Techniques: Northern blot, Hybridization

Journal: Journal of Hematology & Oncology

Article Title: Characterization and validation of potential therapeutic targets based on the molecular signature of patient-derived xenografts in gastric cancer

doi: 10.1186/s13045-018-0563-y

Figure Lengend Snippet: Expressions of several critical molecules in GC PDX models. a EGFR expression was evaluated with scores of: 0, 1+, 2+, and 3+. For the FISH assay, the red and green signals represented EGFR and CEN7, respectively. Scale bar represents 100 μm. b HER3 expression was evaluated with scores of: 0, 1+, 2+, and 3+. Scale bar represents 100 μm. c MET expression was evaluated with H-scores. Scale bar represents 100 μm. d EBV infection status was detected by EBV-encoded RNA in situ hybridization. EBER-positive cells were observed in 20% or more of the tumor cells. Scale bar represents 100 μm. e PD-L1 expression was detected in the placenta (positive control), normal gastric tissue (negative control), and PDX models. Patterns with ≥ 5% positive tumor cells or immune cells was considered to be PDL-1 positive. Scale bar represents 100 μm

Article Snippet: Candidate targets, including EGFR, HER3, MET, and PD-L1, were stained via immunohistochemistry (IHC) using anti-EGFR antibody (#4267, Cell Signaling Technology, Danvers, MA, USA), anti-HER3 antibody (#2708, Cell Signaling Technology), anti-MET antibody (#790-4430, Ventana Medical Systems, Tucson, AZ, USA), and anti-PD-L1 antibody (#M4420, Spring Bioscience Corp., Pleasanton, CA, USA) according to the manufacturers’ instructions.

Techniques: Expressing, Infection, RNA In Situ Hybridization, Positive Control, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Expression Profiling of Ascorbic Acid–Related Transporters in Human and Mouse Eyes

doi: 10.1167/iovs.16-19162

Figure Lengend Snippet: ( a – c ) Immunofluorescence staining of SVCT2 in human and mouse ciliary processes. Sodium-dependent vitamin C transporter 2 expression was limited to the PE layer ( green , arrows ) of the human ciliary process. ( d – f ) Sodium-dependent vitamin C transporter 2 was not identified in the mouse ciliary process. Scale bar : 25 μm.

Article Snippet: The primary antibodies included anti-SVCT2 antibody (1:100; sc-30114; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-GLUT1 antibody (1:100; ab40084; Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Expression Profiling of Ascorbic Acid–Related Transporters in Human and Mouse Eyes

doi: 10.1167/iovs.16-19162

Figure Lengend Snippet: ( a – d ) SLC23A2 (SVCT2) in situ hybridization in human ciliary processes. SVCT2 mRNA (SLC23A2) was detected in the PE layer of human ciliary processes surrounding the nucleus ( red ). Scale bar : 25 μm.

Article Snippet: The primary antibodies included anti-SVCT2 antibody (1:100; sc-30114; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-GLUT1 antibody (1:100; ab40084; Abcam, Cambridge, UK).

Techniques: In Situ Hybridization

Journal: Investigative Ophthalmology & Visual Science

Article Title: Expression Profiling of Ascorbic Acid–Related Transporters in Human and Mouse Eyes

doi: 10.1167/iovs.16-19162

Figure Lengend Snippet: In human retina ( top row ), prominent staining was observed on the basal membrane of the RPE. Sodium-dependent vitamin C transporter 2 expression was also detected in several layers of the neural retina and weakly expressed at the inner segments of PRL, with more intense expression at the OPL and IPL. In mice ( bottom row ), SVCT2 was most intense at the base of segments of PRL, with less staining in the OPL and IPL. Sodium-dependent vitamin C transporter 2 expression in mouse RPE was dispersed and significantly weaker as compared with its PRL. Scale bar : 25 μm.

Article Snippet: The primary antibodies included anti-SVCT2 antibody (1:100; sc-30114; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-GLUT1 antibody (1:100; ab40084; Abcam, Cambridge, UK).

Techniques: Staining, Membrane, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Expression Profiling of Ascorbic Acid–Related Transporters in Human and Mouse Eyes

doi: 10.1167/iovs.16-19162

Figure Lengend Snippet: Colocalization of immunofluorescence staining for SVCT2 (PE in green indicated by arrowhead ) and GLUT1 (NPE in red indicated by arrow ) in human ciliary processes. Sodium-dependent vitamin C transporter 2 and GLUT1 showed clear differences in localization in human ciliary processes. Scale bars : 100 μm and 25 μm.

Article Snippet: The primary antibodies included anti-SVCT2 antibody (1:100; sc-30114; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-GLUT1 antibody (1:100; ab40084; Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Expression Profiling of Ascorbic Acid–Related Transporters in Human and Mouse Eyes

doi: 10.1167/iovs.16-19162

Figure Lengend Snippet: Quantitation of SVCT2 mRNA levels by qPCR in different ocular tissues of humans ( a ) and mice ( b ). Both human and mouse RPE ( red ) are used as references to compare with other ocular tissues. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The primary antibodies included anti-SVCT2 antibody (1:100; sc-30114; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-GLUT1 antibody (1:100; ab40084; Abcam, Cambridge, UK).

Techniques: Quantitation Assay

Journal: The Journal of investigative dermatology

Article Title: Heparin-binding EGF-like growth factor is induced by disruption of lipid rafts and oxidative stress in keratinocytes and participates in the epidermal response to cutaneous wounds.

doi: 10.1038/sj.jid.5701069

Figure Lengend Snippet: Figure 1. p38 MAPK activation and HB-EGF synthesis induced by long-term cholesterol depletion in confluent and postconfluent cultures. (a and b) Keratinocyte cultures were either left untreated or cholesterol- depleted for 18 hours by a combined treatment (1 hour 7.5 mM MBCD/ 17 hours 10 mM lovastatin (lova)) at confluence minus 1 day (C1), confluence (C), or confluence plus one respectively two days (C þ 1 resp. C þ 2). (a) Protein extracts were analyzed by Western blotting using specific antibodies for phospho-p38, p38, involucrin, proHB-EGF, and actin (loading control). The results shown are representative data obtained in three independent experiments. (b) After mRNA extraction, samples were analyzed by Northern blotting using serial hybridizations of the membrane with cDNA probes specific for involucrin and HB-EGF. Gel loading was controlled by hybridization of cDNA specific for the reference gene 36B4. (c) Confluent keratinocyte cultures were treated for 1 hour with 7.5 mM MBCD, 7.5 mM cholesterol (chol), or 7.5 mM MBCD–cholesterol complexes. The data are representative of three independent experiments as analyzed by Western blotting using specific antibodies for phospho-p38 and p38.

Article Snippet: Rabbit anti-human MAPK p38 antibody no. 9212, rabbit anti-human MAPK phospho-p38 Thr180/Tyr182 anti- body no. 9211S, and rabbit anti-human HER-1 antibody no. 2232 were obtained from Cell Signaling (Leiden, The Netherlands).

Techniques: Activation Assay, Western Blot, Control, Extraction, Northern Blot, Membrane, Hybridization

Journal: The Journal of investigative dermatology

Article Title: Heparin-binding EGF-like growth factor is induced by disruption of lipid rafts and oxidative stress in keratinocytes and participates in the epidermal response to cutaneous wounds.

doi: 10.1038/sj.jid.5701069

Figure Lengend Snippet: Figure 2. Lipid raft disruption and oxidative stress induce early p38 activation and subsequent HB-EGF expression during recovery periods. (a–c) Confluent keratinocyte cultures were either left untreated or treated with 7.5 mM MBCD for 1 hour. After the MBCD treatment, the cells were incubated in autocrine culture medium during various periods allowing increasing recovery (recov) times. (d–f) Confluent keratinocyte cultures were either left untreated or subjected to oxidative stress for 20 minutes with 1 mM H2O2. After the applied stress, cells were incubated in autocrine culture medium during various recovery periods. (a and d) Protein extracts were analyzed by Western blotting using specific antibodies for phospho-p38, p38, and proHB-EGF, p38 serving at the same time as a loading control. The results shown are representative data obtained in three independent experiments. (b and e) Illustration of mean relative proHB-EGF expression (normalized to p38)7SD from three independent experiments of which one is shown in (a) and (d), respectively. Statistical analysis was performed by Student’s t-test comparing the values to the expression level of Ctrl for 18 hours (n ¼ 3, *Po0.05; **Po0.01; ***Po0.001). (c and f) Total RNA extracts prepared from three independent cultures were transcribed into cDNA and analyzed as triplicates by real-time PCR to determine relative HB-EGF mRNA expression (normalized to 36B4). Data show the mean relative HB-EGF mRNA expression7SD from three independent experiments. Student’s t-test was performed comparing mRNA levels to mRNA levels of Ctrl for 18 hours (n ¼ 3, *Po0.05; **Po0.01; ***Po0.001).

Article Snippet: Rabbit anti-human MAPK p38 antibody no. 9212, rabbit anti-human MAPK phospho-p38 Thr180/Tyr182 anti- body no. 9211S, and rabbit anti-human HER-1 antibody no. 2232 were obtained from Cell Signaling (Leiden, The Netherlands).

Techniques: Disruption, Activation Assay, Expressing, Incubation, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: The Journal of investigative dermatology

Article Title: Heparin-binding EGF-like growth factor is induced by disruption of lipid rafts and oxidative stress in keratinocytes and participates in the epidermal response to cutaneous wounds.

doi: 10.1038/sj.jid.5701069

Figure Lengend Snippet: Figure 3. HB-EGF synthesis in lipid raft-disrupted keratinocytes is dependent on p38 MAPK. (a) Confluent keratinocyte cultures were pretreated for 30 minutes with 15 mM PD169316, a selective p38 inhibitor, and then cultures were treated for 1 hour with 7.5 mM MBCD the in presence of PD169316, which stayed also present in the recovery medium. Protein extracts were analyzed by Western blotting using specific antibodies for phospho-HSP27, HSP27, phospho-p38, p38, and proHB-EGF. p38 served at the same time as a loading control. The results shown are representative data obtained in three independent experiments. (b) Illustration of mean relative proHB-EGF expression (normalized to p38)7SD from three independent experiments of which one is shown in (a) (n ¼ 3, **Po0.01). (c) Confluent keratinocyte cultures were pretreated with 15 mM PD169316 and then cultures were subjected to long-term cholesterol-depletion (1 hour 7.5 mM MBCD/17 hours 10 mM lova) in the presence of PD169316. Subsequently, RNA messengers were extracted and analyzed by Northern blotting using selective serial hybridization probes for involucrin, HB-EGF, and housekeeping gene 36B4.

Article Snippet: Rabbit anti-human MAPK p38 antibody no. 9212, rabbit anti-human MAPK phospho-p38 Thr180/Tyr182 anti- body no. 9211S, and rabbit anti-human HER-1 antibody no. 2232 were obtained from Cell Signaling (Leiden, The Netherlands).

Techniques: Western Blot, Control, Expressing, Northern Blot, Hybridization

Journal: The Journal of investigative dermatology

Article Title: Heparin-binding EGF-like growth factor is induced by disruption of lipid rafts and oxidative stress in keratinocytes and participates in the epidermal response to cutaneous wounds.

doi: 10.1038/sj.jid.5701069

Figure Lengend Snippet: Figure 7. Epidermal stresses induce HB-EGF expression through p38 MAPK activation in normal human keratinocytes. Stresses like disruption of membrane lipid rafts, oxidative stress, or injury induce HB-EGF expression in a p38-dependent manner in normal human keratinocytes, possibly leading to shedding of HB-EGF into the extracellular environment.

Article Snippet: Rabbit anti-human MAPK p38 antibody no. 9212, rabbit anti-human MAPK phospho-p38 Thr180/Tyr182 anti- body no. 9211S, and rabbit anti-human HER-1 antibody no. 2232 were obtained from Cell Signaling (Leiden, The Netherlands).

Techniques: Expressing, Activation Assay, Disruption, Membrane

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A – C ) Gene expression of Prkaa1 and Prkaa2 (counts per million mapped reads) across different mouse tissues. Box plots show interquartile range, median (line), and minimum and maximum (whiskers). ( A ) Hepatocytes ( n = 4) of mice favor Prkaa2 over Prkaa1 expression while ( B ) macrophages ( n = 8) of mice favor Prkaa1 over Prkaa2 expression. ( C ) Mouse retinas demonstrate appreciable expression levels of Prkaa1 and Prkaa2 ( n = 6). ( D and E ) In situ hybridization of wild-type retina sections confirmed expression of ( D ) Prkaa1 and ( E ) Prkaa2 , seen in magenta dots within the outer nuclear layer (red arrows), suggesting expression of both isoforms in rod photoreceptors. RGC, retinal ganglion cells. ( F ) Scatterplot of Prkaa1 and Prkaa2 expression profiles of mouse retina cell types from single-cell RNA-sequencing data. The cell types show appreciable Prkaa1 and Prkaa2 expression. The rod photoreceptor cluster (red arrow) shows roughly a 2-fold expression of Prkaa2 over Prkaa1 . ( G ) Scatterplot of PRKAA1 and PRKAA2 expression profiles of human retina cell types. The cell types show appreciable PRKAA1 and PRKAA2 expression. The rod photoreceptor cluster (red arrow) shows roughly a 3-fold expression of Prkaa2 over Prkaa1 . ( H ) Scatterplot of Prkaa1 and Prkaa2 expression profiles of mouse brain cell types. Most cell types in the mouse brain demonstrate appreciable expression of Prkaa1 and Prkaa2 . ( I ) Scatterplot of PRKAA1 and PRKAA2 expression profiles of human PBMC types. These cell types do not appreciably express PRKAA2, unlike the central nervous system tissues. ( J ) Waterfall graph depicting the expression ratios of both catalytic isoforms across mouse retina, human retina, mouse brain, and human PBMCs. The majority of neuronal cell types express the α2 isoform over α1 whereas immune cells overwhelmingly express the α1 isoform over α2. Scale bar: 100 μm; insets: 20× original magnification.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Gene Expression, Expressing, In Situ Hybridization, RNA Sequencing

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A and B ) Schematic representations of the protein sequences in the Prkaa1 -Rhod/-Rhod and Prkaa2 -Rhod/-Rhod models, respectively. Knockout domains are within the kinase domain to abrogate function but preserve overall expression of the proteins. ( A ) Schematic representation of the PRKAA1 and ( B ) PRKAA2 protein sequence. ( C ) Representative transmission electron microscopy images of magnification, 2,500×, of rod photoreceptors. (Left) Rod photoreceptors of control mice ( Prkaa2 fl/fl ) demonstrate similar features to those of Prkaa1 -Rhod/-Rhod with consistent membrane structure and organization. (Middle) Rod photoreceptors of Prkaa1 -Rhod/-Rhod demonstrate intact connections between the outer and inner segments with consistent laminar organization of the outer segment membranes. (Right) Rod photoreceptors of Prkaa2 -Rhod/-Rhod consistently demonstrate detachments between the outer and inner segments and occasional outer segment membrane dysmorphisms (red arrow). ( D ) Representative transmission electron microscopy images of magnification, 6,000×, of the outer segments of rod photoreceptors. (Left) Outer segments from control mice ( Prkaa2 fl/fl ) show consistent striations and laminar organization resembling Prkaa1 -Rhod/-Rhod . (Middle) Prkaa1 -Rhod/-Rhod outer segments demonstrated organized and laminar structure indicative of normal wild-type structure. (Right) Prkaa2 -Rhod/-Rhod outer segments exhibited disorganized membrane layers and occasional manifestation of granular debris (red arrows). ( E and F ) Scotopic electroretinography of Prkaa1 -Rhod/-Rhod and respective wild-type littermates. ( E ) Representative traces of 0 dB intensity flashes demonstrate similar waveforms between Prkaa1 fl/fl and Prkaa1 -Rhod/-Rhod ( n = 7). ( F ) Analyses of scotopic a (left) and scotopic b (right) amplitude measurements reveal no significant changes in Prkaa1 -Rhod/-Rhod . ( G and H ) Scotopic electroretinography of Prkaa2 -Rhod/-Rhod and respective wild-type littermates ( n = 7). ( G ) Representative traces of 0 dB intensity flashes show a diminutive waveform from Prkaa2 -Rhod/-Rhod . ( H ) Analyses of scotopic a (left) and scotopic b (right) amplitude measurements confirm significant attenuation in Prkaa2 -Rhod/-Rhod measurements (** P < 0.01, **** P < 0.0001 by 2-way ANOVA with post hoc Bonferroni’s multiple comparisons test). Values are mean ± SEM. Scale bars represent 2 μm. Representative images selected from 40 images for each group.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Knock-Out, Expressing, Sequencing, Transmission Assay, Electron Microscopy, Control, Membrane

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A – H ) Metabolomics analyses of different phospho-purines in Prkaa2 -Rhod/-Rhod retinas using LC-MS/MS ( n = 9). ( A ) Prkaa2 -Rhod/-Rhod retinas demonstrated near-significant increase in cGMP levels, a critical regulator of the dark current ( P = 0.056 by Welch’s t test). ( B and C ) GMP and GDP levels were not significantly changed in Prkaa2 -Rhod/-Rhod . ( D ) GTP levels were significantly increased in Prkaa2 -Rhod/-Rhod (** P < 0.01 by Welch’s t test). ( E ) Levels of IMP, a precursor to GMP, were not significantly changed in Prkaa2 -Rhod/-Rhod . ( F and G ) AMP and ADP levels were not significantly changed in Prkaa2 -Rhod/-Rhod . ( H ) ATP levels were significantly increased in Prkaa2 -Rhod/-Rhod (** P < 0.01 by Welch’s t test). ( I and J ) Extracellular flux analyses by retina Seahorse of Prkaa2 -Rhod/-Rhod . ( I ) Oxygen consumption rate as a measure of oxidative phosphorylative flux was not significantly changed in Prkaa2 -Rhod/-Rhod ( n = 7). ( J ) Extracellular acidification rate as a measure of glycolytic flux was significantly increased ( P < 0.0001 by 2-way ANOVA, comparing the wild-type group to the knockout group as a whole). Glycolysis (* P < 0.05, ** P < 0.01 by post hoc Bonferroni’s multiple comparisons test) and glycolytic capacity (*** P < 0.001 by post hoc Bonferroni’s multiple comparisons test) were significantly upregulated in Prkaa2 -Rhod/-Rhod ( n = 8). ( K ) Excreted retina lactate levels were significantly increased in Prkaa2 -Rhod/-Rhod , supporting the increased glycolysis phenotype ( n = 7, * P < 0.05 by Welch’s t test). Values are mean ± SEM.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Liquid Chromatography with Mass Spectroscopy, Knock-Out

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A ) Schematic representation of the workflow to isolate rod photoreceptors to use for phosphoproteomics analysis. Six retinas were dissected per sample and first dissociated using papain digestion. CD73-PE and Rhodopsin-biotin antibodies were used to tag rod photoreceptor inner and outer segments and were isolated using immunomagnetic beads. The cells were then lysed and processed by LC-MS/MS using an unbiased phosphoproteomics pipeline and analyzed. ( B and C ) Rod photoreceptors from Prkaa1 -Rhod/-Rhod and Prkaa2 -Rhod/-Rhod were processed for phosphoproteomic analyses ( n = 4). 1.75 fold-change and 0.01 P value cutoffs with < 1% false discovery rate were used to determine significant changes. ( B ) Prkaa1 -Rhod/-Rhod analysis revealed 2 downregulated targets, which were unrelated to rod photoreceptor function. ( C ) Prkaa2 -Rhod/-Rhod analysis revealed 45 downregulated targets, including species related to the phototransduction cascade and photoreceptor function. ( D ) A selection of downregulated phosphoproteins from the Prkaa2 -Rhod/-Rhod phosphoproteomics data set were plotted on a heatmap to visualize the spread of individual samples. Samples with higher z scores are visualized as a deeper red color while samples with lower z scores are visualized as a deeper blue color. Each row represents a phospho-site of the denoted protein, while each column represents either a sample from Prkaa2 fl/fl (WT) or Prkaa2 -Rhod/-Rhod (KO). ( E and F ) Tandem mass tag (TMT) signal-to-noise ratios of IMPDH1-S416 and IMPDH2-S416 from individual Prkaa2 fl/fl (WT) and Prkaa2 -Rhod/-Rhod (KO) samples. KO samples are colored as pink dots while WT samples are colored as blue dots. In both IMPDH1-S416 and IMPDH2-S416 measurements, the KO samples overall present lower signal-to-noise ratios than WT samples.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Phospho-proteomics, Isolation, Liquid Chromatography with Mass Spectroscopy, Selection

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A ) Schematic representation of IMPDH function in rod photoreceptors according to light exposure. Previous work has shown GTP allosterically inhibits IMPDH in dark-adapted conditions while IMPDH is active to produce GMP in light-adapted conditions. ( B ) Ambient light–adapted retinas from Prkaa2 -Rhod/-Rhod were assessed for IMPDH activity ( n = 4). No significant changes were detected. ( C ) Dark-adapted retinas from Prkaa2 -Rhod/-Rhod showed significantly increased IMPDH activity ( n = 6; ** P < 0.01 by 2-way ANOVA). ( D ) Ambient light–adapted wild-type retinas were assessed for IMPDH activity after treatment with 5 mM metformin, an AMPK activator. Metformin is a slower activator of AMPK, and metformin treatment inhibits IMPDH activity compared with that of control retinas ( n = 6, P < 0.0001 by 2-way ANOVA). ( E ) Dark-adapted wild-type retinas were assessed for IMPDH activity after treatment with 40 μM compound C, an AMPK inhibitor. Lysates treated with compound C exhibited significantly higher IMPDH activity compared with controls ( n = 4, ** P < 0.01 by 2-way ANOVA). ( F and G ) Recombinant human AMPK (α2β1γ1) was incubated with recombinant human ( F ) IMPDH1 or ( G ) IMPDH2 to assess the effect of AMPK on IMPDH activity. Both IMPDH1 and IMPDH2 when incubated with AMPK had significantly attenuated IMPDH activity compared with IMPDH incubated alone ( n = 4, P < 0.0001 by 2-way ANOVA). ( H ) Schematic workflow of the co-immunoprecipitation protocol of IMPDH1. Six dark-adapted retinas from wild-type mice were dissected and lysed in extraction buffer. Dynabeads coated with IMPDH1 antibody were added to the suspension and allowed to bind to IMPDH1. Attached IMPDH1 along with bound proteins were isolated. The resulting eluant was then used for Western blots to detect IMPDH1 and bound protein. ( I ) The results of Western blot detection of co-immunoprecipitation samples are depicted. Clear bands representing IMPDH1 along with PRKAA2 demonstrate PRKAA2 was bound to IMPDH1 in wild-type retinas. Values are mean ± SEM.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: Activity Assay, Control, Recombinant, Incubation, Immunoprecipitation, Extraction, Suspension, Isolation, Western Blot

Journal: JCI Insight

Article Title: Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

doi: 10.1172/jci.insight.173707

Figure Lengend Snippet: ( A and B ) Representative electroretinography traces of Prkaa2 -Rhod/-Rhod mass rod recovery. A 0 dB flash with 800 ms interstimulus time (IST) is represented. ( A ) A trace from Prkaa2 fl/fl shows typical attenuated scotopic a and b waves in the test response indicated by the probe flash with 800 ms interstimulus time. ( B ) A trace from Prkaa2 -Rhod/-Rhod shows abnormally large scotopic a and b waves with 800 ms IST in the test response indicated by the probe flash. ( C ) Quantification of scotopic A and scotopic B rod recovery ( n = 7). Prkaa2 -Rhod/-Rhod mice show significantly faster rod recovery compared to wild-type littermates (*** P < 0.001, **** P < 0.0001 by post hoc Bonferroni’s multiple comparisons test; P < 0.0001 by 2-way ANOVA for both graphs). ( D and E ) Representative electroretinography traces of Prkaa2 -Rhod/-Rhod mass rod recovery from eyes treated with vehicle or mycophenolate mofetil. ( D ) Vehicle-treated eyes demonstrate similar waveform shapes as ( E ) mycophenolate-treated eyes from the probe flash. ( F ) Quantification of scotopic a and scotopic b rod recovery ( n = 5). Mycophenolate-treated eyes show significantly slower scotopic a and scotopic b rod recovery compared with vehicle-treated eyes ( †† P < 0.01 by post hoc Bonferroni’s multiple comparisons test; *** P < 0.001 by 2-way ANOVA). ( G ) Representative traces of full-field scotopic electroretinography from vehicle- and mycophenolate-treated eyes. Although the waveform shape is slightly altered, mycophenolate treatment improves scotopic a and scotopic b wave amplitudes compared with vehicle treatment. ( H ) Quantification of scotopic a and scotopic b wave amplitudes from full-field scotopic electroretinography ( n = 5). Mycophenolate-treated eyes had significantly improved scotopic a and scotopic b wave amplitudes compared with vehicle-treated eyes (** P < 0.01, *** P < 0.001 by 2-way ANOVA). Values are mean ± SEM.

Article Snippet: IMPDH1 (Proteintech catalog 22092-1-AP) and PRKAA2 (R&D Systems catalog AF2850) were used for primary antibody incubation.

Techniques: