Journal: BMC Neuroscience

Article Title: PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration

doi: 10.1186/s12868-018-0479-z

Figure Lengend Snippet: PKCε phosphorylation of ATF2 at T52 in vivo. a Representative Western blot showing that anti-phospho-ATF2 (T52) antibody detected less immunoreactivity in hippocampal lysates from Prkce − / − than Prkce + / + mice. b The ratio of phospho-ATF2 (T52) to β-Actin immunoreactivity was significantly reduced in the lysates from Prkce − / − ( n = 3) as compared to Prkce + / + mice ( n = 3) (* P < 0.05, two-tailed, unpaired t -test). c The ratio of ATF2 to β-Actin immunoreactivity was similar between Prkce + / + ( n = 3) and Prkce − / − mice ( n = 3). Error bars show SEM

Article Snippet: The whole cell lysates, mitochondrial and cytosolic fractions were analyzed by Western blot using rabbit anti-Thr-52 ATF2 (1:200, Phosphosolution, Aurora, CO, USA, Cat# p115-52), rabbit anti-ATF2 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9226), mouse anti-Actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA, Cat# A4700), mouse anti-PKCε (1:1000, BD, Franklin Lakes, NJ, USA, Cat# 610086) and rabbit anti-COXIV (1:1000, Sigma-Aldrich, Cat# AV42784).

Techniques: Phospho-proteomics, In Vivo, Western Blot, Two Tailed Test

Journal: BMC Neuroscience

Article Title: PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration

doi: 10.1186/s12868-018-0479-z

Figure Lengend Snippet: Temporal expression of ATF2 in the hippocampal mitochondria after tGCI. a Mitochondrial fractions of hippocampus isolated at different time points after tGCI were analyzed by Western blotting using antibodies against ATF2. COXIV was used as a loading control for the mitochondria. b Quantitative analysis of ATF2/COXIV protein bands by densitometry demonstrated a significant induction of mitochondrial ATF2 expression 24 and 48 h after tGCI as compared with controls ( n = 4–5) (*** p < 0.0005, one-way ANOVA with Newman-Keuls post hoc tests)

Article Snippet: The whole cell lysates, mitochondrial and cytosolic fractions were analyzed by Western blot using rabbit anti-Thr-52 ATF2 (1:200, Phosphosolution, Aurora, CO, USA, Cat# p115-52), rabbit anti-ATF2 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9226), mouse anti-Actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA, Cat# A4700), mouse anti-PKCε (1:1000, BD, Franklin Lakes, NJ, USA, Cat# 610086) and rabbit anti-COXIV (1:1000, Sigma-Aldrich, Cat# AV42784).

Techniques: Expressing, Isolation, Western Blot, Control

Journal: BMC Neuroscience

Article Title: PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration

doi: 10.1186/s12868-018-0479-z

Figure Lengend Snippet: Mitochondrial translocation of ATF2 after oxygen–glucose–deprivation. Confocal images show the expression pattern of ATF2 (green) and MitoTracker (red) in cultured hippocampal neurons under non-ischemic conditions ( a ) and 4 h after oxygen–glucose–deprivation ( b ). Nuclei were stained with DAPI (blue). Scale bars, 10 μm

Article Snippet: The whole cell lysates, mitochondrial and cytosolic fractions were analyzed by Western blot using rabbit anti-Thr-52 ATF2 (1:200, Phosphosolution, Aurora, CO, USA, Cat# p115-52), rabbit anti-ATF2 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9226), mouse anti-Actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA, Cat# A4700), mouse anti-PKCε (1:1000, BD, Franklin Lakes, NJ, USA, Cat# 610086) and rabbit anti-COXIV (1:1000, Sigma-Aldrich, Cat# AV42784).

Techniques: Translocation Assay, Expressing, Cell Culture, Staining

Journal: BMC Neuroscience

Article Title: PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration

doi: 10.1186/s12868-018-0479-z

Figure Lengend Snippet: Mitochondrial translocation of ATF2 after tGCI. Confocal images of ATF2 (red) and COXIV (green) in the CA1 of the hippocampus under non-ischemic conditions ( a – d , i – l ), 24 ( e – h , m – p ) and 48 h ( q – t ) after tGCI. Amplified images are shown in the right panels ( i – t ). Merged images ( d , h , l , p , t ) indicate colocalization of ATF2 with COXIV (yellow) after tGCI (white arrowheads). Scale bars, 50 μm

Article Snippet: The whole cell lysates, mitochondrial and cytosolic fractions were analyzed by Western blot using rabbit anti-Thr-52 ATF2 (1:200, Phosphosolution, Aurora, CO, USA, Cat# p115-52), rabbit anti-ATF2 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9226), mouse anti-Actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA, Cat# A4700), mouse anti-PKCε (1:1000, BD, Franklin Lakes, NJ, USA, Cat# 610086) and rabbit anti-COXIV (1:1000, Sigma-Aldrich, Cat# AV42784).

Techniques: Translocation Assay, Amplification

Journal: BMC Neuroscience

Article Title: PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration

doi: 10.1186/s12868-018-0479-z

Figure Lengend Snippet: Expression of ATF2 in degenerating neurons labeled with Fluoro-Jade C. Confocal images of ATF2 (red) and Fluoro-Jade C (green) in the CA1 of the hippocampus under non-ischemic conditions ( a – d ) and 48 h after tGCI ( e – h ). Merged images ( d , h ) indicate colocalization of ATF2 with Fluoro-Jade C (yellow) after tGCI ( h ). Neurons with cytoplasmic expression of ATF2 (arrowheads) and induced expression of ATF2 (arrows) were indicated in panel E-H. Scale bars, 25 μm

Article Snippet: The whole cell lysates, mitochondrial and cytosolic fractions were analyzed by Western blot using rabbit anti-Thr-52 ATF2 (1:200, Phosphosolution, Aurora, CO, USA, Cat# p115-52), rabbit anti-ATF2 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9226), mouse anti-Actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA, Cat# A4700), mouse anti-PKCε (1:1000, BD, Franklin Lakes, NJ, USA, Cat# 610086) and rabbit anti-COXIV (1:1000, Sigma-Aldrich, Cat# AV42784).

Techniques: Expressing, Labeling

Journal: BMC Neuroscience

Article Title: PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration

doi: 10.1186/s12868-018-0479-z

Figure Lengend Snippet: A proposed model for PKCε phosphorylation of ATF2 during ischemia-induced neurodegeneration. Phosphorylation of ATF2 by PKCε at Thr-52 is required for the nuclear localization of ATF2. The expression level of PKCε is attenuated in response to global cerebral ischemia, resulting in translocation of ATF2 to the mitochondria and degeneration of neuronal cells

Article Snippet: The whole cell lysates, mitochondrial and cytosolic fractions were analyzed by Western blot using rabbit anti-Thr-52 ATF2 (1:200, Phosphosolution, Aurora, CO, USA, Cat# p115-52), rabbit anti-ATF2 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9226), mouse anti-Actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA, Cat# A4700), mouse anti-PKCε (1:1000, BD, Franklin Lakes, NJ, USA, Cat# 610086) and rabbit anti-COXIV (1:1000, Sigma-Aldrich, Cat# AV42784).

Techniques: Phospho-proteomics, Expressing, Translocation Assay

Journal: Molecular and Cellular Biology

Article Title: Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

doi: 10.1128/mcb.01344-08

Figure Lengend Snippet: FIG. 1. Potassium deprivation induces the transactivation of c-Jun and ATF2 and the downregulation of c-Fos. (A) CGNs were cultured in 25 K or 5 K medium for the indicated times, and cell lysates were subjected to Western blotting with antibodies against phospho-c-Jun (p-c-Jun), c-Jun, phospho-ATF2, or ATF2. (B) CGNs in 25 K or 5 K medium were treated with 10 M SP600125 (SP) or 2 M CEP11004 (CEP) for 4 h, and cell lysates were subjected to Western blotting (WB) with antibodies against phospho-ATF2 or ATF2. (C) CGNs in 25 K or 5 K medium were lysed at the times indicated and subjected to Western blotting with antibodies against c-Fos. Total mRNA was extracted and subjected to RT-PCR with primers specific to c-fos. (D) CGNs were cultured in 25 K or 5 K medium for 4 h in the presence or absence of 1 M nifedipine (Nif) or 10 M KN62 (KN). Lysates were then subjected to Western blotting with antibodies against c-Fos. Total mRNA was extracted and subjected to RT-PCR with primers specific to c-fos. Tubulin was reprobed to verify equal loadings, and -actin was amplified to verify equal inputs.

Article Snippet: Primary antibodies included phospho-ATF2 (Thr69 Thr71), phospho-c-Jun (Ser73) (Cell Signaling Technology), ATF2, c-Fos (Santa Cruz Biotechnology), c-Jun (BD Bioscience), tubulin, Flag (Sigma), and V5 tag antibody (AbD Serotec).

Techniques: Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

doi: 10.1128/mcb.01344-08

Figure Lengend Snippet: FIG. 2. The majority of c-Jun heterodimerizes with ATF2, and c-Jun/ATF2 heterodimers predominantly occupy the ATF sites in CGNs deprived of potassium. (A) CGNs in 25 K or 5 K medium for 4 h were lysed and subjected to IP with either ATF2 or c-Jun antibodies. The precipitates were analyzed by Western blotting (WB). (B) Cell lysates from CGNs incubated in either 25 K or 5 K medium for 4 h were subjected to immunodepletion (ID) using ATF2 or c-Fos antibodies. The supernatants were analyzed by Western blotting with a c-Jun antibody. The intensity of the c-Jun band was normalized to tubulin. (C) Gel mobility shift assays were performed using 32P-labeled atf probes or dp5 probes with nuclear extracts depleted of c-Jun or ATF2. Immunoglobulin G (IgG) was used as an antibody control. SB, specific DNA-protein band.

Article Snippet: Primary antibodies included phospho-ATF2 (Thr69 Thr71), phospho-c-Jun (Ser73) (Cell Signaling Technology), ATF2, c-Fos (Santa Cruz Biotechnology), c-Jun (BD Bioscience), tubulin, Flag (Sigma), and V5 tag antibody (AbD Serotec).

Techniques: Western Blot, Incubation, Immunodepletion, Mobility Shift, Labeling, Control

Journal: Molecular and Cellular Biology

Article Title: Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

doi: 10.1128/mcb.01344-08

Figure Lengend Snippet: FIG. 3. Potassium deprivation increases ATF activity rather than TRE activity. (A) CGNs were transfected with atf-luc plasmids and pCMV-RL in combination with plasmids encoding c-Jun–DN, ATF2- DN, or A-ATF2 (left panel). A dual-reporter analysis of dp5-luc or atf3-luc was performed with CGNs incubated in either 25 K or 5 K medium (right panel). The levels of luciferase activity were normalized to Renilla luciferase activity. (B) The levels of c-jun, dp5, and atf3 mRNA in CGNs in 25 K or 5 K medium for the indicated durations of time were measured, and -actin mRNA was amplified as an input control. (C) Dual-reporter analysis of tre-luc or coll-luc in CGNs cultured in 25 K or 5 K medium. Data in this figure are presented as means standard errors of the means (SE) (three experiments). P 0.05 (Student’s t test).

Article Snippet: Primary antibodies included phospho-ATF2 (Thr69 Thr71), phospho-c-Jun (Ser73) (Cell Signaling Technology), ATF2, c-Fos (Santa Cruz Biotechnology), c-Jun (BD Bioscience), tubulin, Flag (Sigma), and V5 tag antibody (AbD Serotec).

Techniques: Activity Assay, Transfection, Incubation, Luciferase, Control, Cell Culture

Journal: Molecular and Cellular Biology

Article Title: Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

doi: 10.1128/mcb.01344-08

Figure Lengend Snippet: FIG. 4. c-Jun/ATF2 heterodimers are necessary and sufficient for CGN apoptosis. (A) CGNs transfected with c-Jun–DN, ATF2-DN, or A-ATF2 together with GFP (to mark the transfected cells) were maintained in 25 K or 5 K medium for 12 h and stained with Hoechst 33258. (B) HEK 293 cells were cotrans- fected with the constructs BS/U6, shc-juna, or shc-junb together with Flag–c-Jun and subjected to Western blotting with a Flag antibody (left panel). CGNs transfected with BS/U6, shc-juna, or shc-junb plasmids were maintained in 25 K or 5 K medium and then subjected to c-Jun staining, with -Gal staining as a marker for transfection (middle panel) or with GFP to analyze apoptosis (right panel). Scale bar 10 m. (C) Knockdown efficiency and apoptotic analysis for shatf2a and shatf2b were performed as described for panel B. (D) HEK 293 cells were transfected with V5-C2/c-Jun and Flag-C2/ATF2 and then lysed for CoIP with V5 or Flag antibodies. Western blotting (WB) was performed using a Flag or V5 antibody. (E) CGNs were cotransfected with C2/c-Jun and C2/ATF2 in the indicated doses and in the absence or presence of atf-luc plasmids (left panel). A dual-reporter analysis was performed. An apoptotic analysis was performed 48 h posttransfection. All apoptotic rates in this figure were quantified by scoring the percentages of transfected neurons with pyknotic nuclei in total transfected cells. The data are presented as means SE of the means (three experiments). , P 0.05; , P 0.01 (Student’s t test).

Article Snippet: Primary antibodies included phospho-ATF2 (Thr69 Thr71), phospho-c-Jun (Ser73) (Cell Signaling Technology), ATF2, c-Fos (Santa Cruz Biotechnology), c-Jun (BD Bioscience), tubulin, Flag (Sigma), and V5 tag antibody (AbD Serotec).

Techniques: Transfection, Staining, Construct, Western Blot, Marker, Knockdown

Journal: Molecular and Cellular Biology

Article Title: Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

doi: 10.1128/mcb.01344-08

Figure Lengend Snippet: FIG. 5. Blocking c-Jun/ATF2 heterodimers using decoy oligonucle- otides suppresses ATF activity and apoptosis. CGNs transfected with the atf decoy (atf-dec) or mutant atf decoy (m-atf-dec) together with atf-luc were maintained in 25 K or 5 K medium and were subjected to dual-reporter analysis. Transfection of either the atf decoy or the mutant atf decoy alone as well as apoptotic analyses of the cells were performed as described in the legend to Fig. 4A. Data are presented as means SE (three experiments). P 0.05 (Student’s t test).

Article Snippet: Primary antibodies included phospho-ATF2 (Thr69 Thr71), phospho-c-Jun (Ser73) (Cell Signaling Technology), ATF2, c-Fos (Santa Cruz Biotechnology), c-Jun (BD Bioscience), tubulin, Flag (Sigma), and V5 tag antibody (AbD Serotec).

Techniques: Blocking Assay, Activity Assay, Transfection, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

doi: 10.1128/mcb.01344-08

Figure Lengend Snippet: FIG. 6. c-Fos downregulation facilitates c-Jun/ATF2 heterodimerization in response to potassium deprivation in CGNs. (A) CGNs transfected with plasmids encoding JunVN173 (JunVN) and ATF2VC155 (ATF2VC), in combination with plasmids encoding CFP, were maintained in 25 K or 5 K medium for 1 h, and photographs were obtained, or cells were lysed for Western blotting to determine the expression levels of JunVN and ATF2VC. JunL3VN served as a control. The white arrows indicate BiFC signals (Venus fluorescence). The percentage of CFP-positive cells exhibiting Venus fluorescence was scored. Data are presented as means SE (three experiments). P 0.05 (Student’s t test). (B) CGNs cotransfected with plasmids encoding c-Fos or A-Fos together with plasmids encoding JunVN, ATF2VC, and CFP were subjected to BiFC analysis. Data are presented as means SE (three experiments). P 0.05 (Student’s t test). (C) HEK 293 cells cotransfected with BS/U6, shc-fosa, or shac-fosb together with plasmids encoding Flag–c-Fos were subjected to Western blotting with a Flag antibody. CGNs were transfected with the BS/U6, shc-fosa, or shc-fosb plasmids together with plasmids encoding JunVN, ATF2VC, and CFP for BiFC analysis. Data are presented as means SE (three experiments). P 0.05 (Student’s t test).

Article Snippet: Primary antibodies included phospho-ATF2 (Thr69 Thr71), phospho-c-Jun (Ser73) (Cell Signaling Technology), ATF2, c-Fos (Santa Cruz Biotechnology), c-Jun (BD Bioscience), tubulin, Flag (Sigma), and V5 tag antibody (AbD Serotec).

Techniques: Transfection, Western Blot, Expressing, Control

Journal: Molecular and Cellular Biology

Article Title: Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

doi: 10.1128/mcb.01344-08

Figure Lengend Snippet: FIG. 7. Overexpression of c-Fos or A-Fos attenuates c-Jun/ATF2 heterodimer-mediated ATF activity, expression of target genes, and apoptosis. (A) CGNs transfected with plasmids encoding c-Fos or A-Fos together with atf-luc were maintained in 25 K or 5 K medium and subjected to a dual-reporter assay. (B) HEK 293 cells cotransfected with V5-C2/c-Jun and Flag-C2/ATF2 with or without c-Fos or A-Fos were lysed for IP with a V5 antibody. Input and precipitates were analyzed by Western blotting (WB) (left panel). CGNs were transfected with atf-luc plasmids together with plasmids encoding C2/ATF2, C2/c-Jun, c-Fos, and A-Fos and subjected to a dual-reporter assay (right panel). (C) CGNs infected with Ad–c-Fos or Ad–A-Fos were stained with a Flag antibody or not stained with a primary antibody ( 1°) (left panel). Western blotting was performed to detect the expression of c-Fos or A-Fos (right panel). (D) Cell lysates were subjected to a ChIP assay with c-Jun or ATF2 antibodies. Chromatin was sonicated into fragments of approximately 500 bp (left panel). Western blotting analysis of the precipitates was performed using c-Jun or ATF2 antibodies (middle panel). Purified chromatin was assessed by PCR amplification of the regions proximal to the c-jun, dp5, and atf3 promoters (right panel). Equal amounts of total genomic DNA (Input) were used for IP under each condition. (E) CGNs infected with Ad-GFP, Ad–c-Fos, or Ad–A-Fos were incubated in 25 K or 5 K medium and subjected to RT-PCR with primers specific to c-jun, dp5, atf3, and bim. -actin was amplified to verify equal inputs. (F) CGNs infected with Ad-GFP, Ad–c-Fos, or Ad–A-Fos were maintained in 25 K or 5 K medium for apoptotic analysis. Data were presented as means SE (three experiments). P 0.05 (Student’s t test).

Article Snippet: Primary antibodies included phospho-ATF2 (Thr69 Thr71), phospho-c-Jun (Ser73) (Cell Signaling Technology), ATF2, c-Fos (Santa Cruz Biotechnology), c-Jun (BD Bioscience), tubulin, Flag (Sigma), and V5 tag antibody (AbD Serotec).

Techniques: Over Expression, Activity Assay, Expressing, Transfection, Reporter Assay, Western Blot, Infection, Staining, Sonication, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis

doi: 10.1128/mcb.01344-08

Figure Lengend Snippet: FIG. 8. Model for the signaling pathways regulating potassium dep- rivation-induced CGN apoptosis. Deprivation of the survival agent, potassium, not only results in JNK-dependent transactivation of c-Jun and ATF2 via phosphorylation of their N-terminal regions but also leads to concurrent c-Fos downregulation attributable to the decreased activity of Ca2/calmodulin (CaM) and CaMKs secondary to a loss of calcium influx. c-Jun primarily heterodimerizes with ATF2, and c-Jun/ ATF2 heterodimers are responsible for the upregulation of ATF ac- tivity. c-Fos downregulation facilitates the heterodimerization between c-Jun and ATF2. The upregulation of ATF activity triggers target gene expression (dp5, atf3, and other genes) to promote neuronal apoptosis. p-c-Jun, phospho-c-Jun.

Article Snippet: Primary antibodies included phospho-ATF2 (Thr69 Thr71), phospho-c-Jun (Ser73) (Cell Signaling Technology), ATF2, c-Fos (Santa Cruz Biotechnology), c-Jun (BD Bioscience), tubulin, Flag (Sigma), and V5 tag antibody (AbD Serotec).

Techniques: Protein-Protein interactions, Phospho-proteomics, Activity Assay, Targeted Gene Expression