Journal: bioRxiv

Article Title: The human mitochondrial 12S rRNA m 4 C methyltransferase METTL15 is required for proper mitochondrial function

doi: 10.1101/809756

Figure Lengend Snippet: (A) Comparison of mitochondrial genome (mtDNA) copy number between the Control and METTL15 KO cells, measured by RT-qPCR. (B) Expression analysis by RT-qPCR of mitochondrial coding genes in the control and METTL15 KO cells. (C) The protein level of COX2 is downregulated in successfully edited cell lines (red fonts) but not changed in parental or unedited cells (black fonts). (D) Western blot analysis of the indicated proteins in the control, METTL15 KO, and METTL15 KO cells rescued with the wildtype or catalytic mutant of METTL15-Flag-HA, respectively. LMNB1 and Actin were used as controls.

Article Snippet: Anti-COX2 (55070-1-AP)antibody was purchased from Proteintech.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: The human mitochondrial 12S rRNA m 4 C methyltransferase METTL15 is required for proper mitochondrial function

doi: 10.1101/809756

Figure Lengend Snippet: (A) The distribution of the mitochondrial ribosome small and large subunits in the indicated sucrose gradient fractions, examined by 12S and 16S rRNA RT-qPCR. (B) The distribution of the mRNAs of the mitochondrial coding genes in the indicated sucrose gradient fractions examined by RT-qPCR. (C) Western blot analyses of ND6 and COX2 protein levels in the control and METTL15 KO cells. The bar graph represents the quantification results of 2 replicate experiments.

Article Snippet: Anti-COX2 (55070-1-AP)antibody was purchased from Proteintech.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Endocrine-Related Cancer

Article Title: Anti-inflammatory therapies in TRAMP mice: delay in PCa progression

doi: 10.1530/erc-15-0540

Figure Lengend Snippet: Figure 7. Western blotting analysis of cyclooxygenase-2 (COX2), phosphorylated signal transducer and activator of transcription 3 (pSTAT3), factor nuclear kappa B (NFkB), insulin-like growth factor receptor 1 (IGFR1) protein levels in the ventral prostate of the experimental groups. Different lowercase letters indicate

Article Snippet: The COX2, STAT3, 159 IGFR1, antigens were detected, respectively, using the following antibodies: mouse 160 monoclonal anti COX2 (sc-376861- Santa Cruz Biotechnology, Santa Cruz, CA, United 161 States of America), rabbit polyclonal anti-STAT3 (sc-7179 - Santa Cruz Biotechnology, 162 Santa Cruz, CA, USA), rabbit polyclonal anti- IGFR1 (sc-712- Santa Cruz Biotechnology, 163 Santa Cruz, CA, USA), mouse monoclonal anti-PCNA (ab-29 – Abcam, Cambridge, MA, 164 USA).

Techniques: Western Blot

Journal: Endocrine-Related Cancer

Article Title: Anti-inflammatory therapies in TRAMP mice: delay in PCa progression

doi: 10.1530/erc-15-0540

Figure Lengend Snippet: Figure 8. COX2, STAT3, IGFR1 immunoreactivities in the ventral prostate of TRAMP mice from T8 (A, E, I), T12 (B, F, J) T1GTN (C, G, K) and T1CEL (D, H, L). Epithelial and stromal reactivities were graded according

Article Snippet: The COX2, STAT3, 159 IGFR1, antigens were detected, respectively, using the following antibodies: mouse 160 monoclonal anti COX2 (sc-376861- Santa Cruz Biotechnology, Santa Cruz, CA, United 161 States of America), rabbit polyclonal anti-STAT3 (sc-7179 - Santa Cruz Biotechnology, 162 Santa Cruz, CA, USA), rabbit polyclonal anti- IGFR1 (sc-712- Santa Cruz Biotechnology, 163 Santa Cruz, CA, USA), mouse monoclonal anti-PCNA (ab-29 – Abcam, Cambridge, MA, 164 USA).

Techniques:

Journal: Endocrine-Related Cancer

Article Title: Anti-inflammatory therapies in TRAMP mice: delay in PCa progression

doi: 10.1530/erc-15-0540

Figure Lengend Snippet: Figure 9. COX2, STAT3, IGFR1 immunoreactivities in the ventral prostate of TRAMP mice from T22 (A, D, G,), T2GTN (B, E, H) and T2CEL (C, F, I). Epithelial and stromal reactivities were graded according to Table

Article Snippet: The COX2, STAT3, 159 IGFR1, antigens were detected, respectively, using the following antibodies: mouse 160 monoclonal anti COX2 (sc-376861- Santa Cruz Biotechnology, Santa Cruz, CA, United 161 States of America), rabbit polyclonal anti-STAT3 (sc-7179 - Santa Cruz Biotechnology, 162 Santa Cruz, CA, USA), rabbit polyclonal anti- IGFR1 (sc-712- Santa Cruz Biotechnology, 163 Santa Cruz, CA, USA), mouse monoclonal anti-PCNA (ab-29 – Abcam, Cambridge, MA, 164 USA).

Techniques:

Journal: Inflammation research : official journal of the European Histamine Research Society ... [et al.]

Article Title: Evidence that α -lipoic acid inhibits NF- κ B activation independent of its antioxidant function

doi: 10.1007/s00011-010-0256-7

Figure Lengend Snippet: The antioxidant function of LA is not involved in its inhibition of TNFα-induced NF-κB activation. a, b HUVECs were starved overnight and pre-treated with various concentration of LA for 20 min, followed by treatment with TNFα (10 ng/ml) for 10 min. Cells were then lyzed in RIPA buffer, and IκBα was analyzed by Western blotting. A representative picture (a) and the summary (b) of three independent experiments are presented. *P < 0.05 versus TNFα only, Student's t-test with Bonferoni correction. c, d HUVECs were starved overnight and pre-treated with LA (1 mM), tiron (20 mM), apocynin (0.3 mM), PEG-SOD (100 U/ml) and tempol (0.1 mM) for 20 min, followed by treatment with TNFα (10 ng/ml) for 10 min. Cells were then lysed in RIPA buffer, and IκBα was analyzed by Western blotting. A representative picture (c) and the summary (d) of four independent experiments are presented. *P < 0.05 versus vehicle, Student's t-test with Bonferoni correction. e, f HUVECs were starved overnight and pre-treated with LA (1 mM), tiron (20 mM), apocynin (0.3 mM), PEG-SOD (100 U/ml) and tempol (0.1 mM) for 20 min, followed by treatment with TNFα (10 ng/ml) for 30 min (NF-κB DNA binding activity assay) or 8 h (all other measurements). Nuclear proteins or total proteins were prepared. The NF-κB DNA binding activity was assessed by EMSA, and a representative results of two independent experiments is presented. The expression of COX2 and VCAM-1 were examined by Western blot. A representative picture (e) and the summary (f) of three independent experiments were presented. *P < 0.05 versus vehicle, Student's t-test with Bonferoni correction. g In the presence of the indicated antioxidants, the superoxide production by NADPH oxidase in mouse liver lysate was measured by lucigenin chemiluminescence assay, n = 6, *P < 0.05 versus control, one way-ANOVA

Article Snippet: Western blotting This was performed using standard techniques with primary antibodies as follows: monoclonal anti- β -actin (Sigma), rabbit anti-I κ B α (Upstate Biotechnology), monoclonal anti-vascular cell adhesion molecule-1 (VCAM-1) (R&D Systems), monoclonal anti-cyclo-oxygenase-2 (COX2) (Cayman Chemical), goat anti-Akt1 (Santa Cruz), and rabbit anti-phospho-Akt (Cell Signaling Technology).

Techniques: Inhibition, Activation Assay, Concentration Assay, Western Blot, Binding Assay, Activity Assay, Expressing, Chemiluminescence Immunoassay

Journal: Inflammation research : official journal of the European Histamine Research Society ... [et al.]

Article Title: Evidence that α -lipoic acid inhibits NF- κ B activation independent of its antioxidant function

doi: 10.1007/s00011-010-0256-7

Figure Lengend Snippet: LA inhibits IKK2 in living cells. a HEK293 cells were transfected with a constitutively active mutant of IKK2, IKK2(EE), and a NF-κB reporter construct. Twenty-four hours later, these cells were treated with the indicated concentration of LA for 24 h, and the activity of the reporter gene, luciferase, was analyzed by chemiluminescence assay, n = 3. b and c HEK293 cells were transfected with a constitutively active mutant of IKK2, IKK2(EE), and a NF-κB reporter construct. Twenty-four hours later, these cells were treated with the indicated concentration of LA for 24 h, and the expression of COX2 was analyzed by Western blot, n = 3. *P < 0.05 versus IKK2(EE)−/LA−, #P < 0.05 versus IKK2(EE)+/LA−, Student's t-test with Bonferoni correction

Article Snippet: Western blotting This was performed using standard techniques with primary antibodies as follows: monoclonal anti- β -actin (Sigma), rabbit anti-I κ B α (Upstate Biotechnology), monoclonal anti-vascular cell adhesion molecule-1 (VCAM-1) (R&D Systems), monoclonal anti-cyclo-oxygenase-2 (COX2) (Cayman Chemical), goat anti-Akt1 (Santa Cruz), and rabbit anti-phospho-Akt (Cell Signaling Technology).

Techniques: Transfection, Mutagenesis, Construct, Concentration Assay, Activity Assay, Luciferase, Chemiluminescence Immunoassay, Expressing, Western Blot

Journal: British Journal of Cancer

Article Title: Reversal of gene expression changes in the colorectal normal-adenoma pathway by NS398 selective COX2 inhibitor

doi: 10.1038/sj.bjc.6605515

Figure Lengend Snippet: The decrease in COX2 protein expression under NS398 treatment. Dose-dependent inhibition of COX2 protein expression was observed under NS398 treatment. Strong granular and/or diffuse cytoplasmatic immunostaining was detected in COX2-positive cells. ( A ) COX2 protein expression in untreated control HT29 cells. ( B ) COX2 protein expression in HT29 cells treated with 100 μ M NS398 ( × 10 magnification, haematoxylin co-staining). ( C ) COX2 protein expression detected by western blot analysis. The diagram shows the band intensities determined by Molecular Imaging Software version 4.0.

Article Snippet: Western blot analysis of COX2 (rabbit anti-human polyclonal COX2 antibody, Code: RB-9072, 1 μ g ml −1 , Thermo Fisher Scientific) was performed as previously described ( Tátrai et al , 2006 ).

Techniques: Expressing, Inhibition, Immunostaining, Control, Staining, Western Blot, Imaging, Software

Journal: Aging (Albany NY)

Article Title: Alterations in oxidative, inflammatory and apoptotic events in short-lived and long-lived mice testes

doi:

Figure Lengend Snippet: Panel ( A ) COX2 (72 kDa) expression was evaluated by immunoblotting in testicular homogenates of short-lived (GH-Tg) and long-lived (GHRH-KO and Ames dwarf) mice. Bar plot graphs represent the mean ± S.E.M. and depict the quantification by densitometry of the bands. Results were normalized to actin (42 kDa) and expressed as fold change relative to the control (normal littermates), which was assigned a value of 1. Bar plot graphs represent the mean + SEM; n = 5-7. * p < 0.05, t-Student test. Panel ( B ) PGD2 testicular levels were determined by immunoassay in testicular homogenates from short-lived (GH-Tg) and long-lived (GHRH-KO and Ames dwarf) mice. Bar plot graphs represent the mean + SEM; n = 5-7. * p < 0.05, t-Student test.

Article Snippet: Testicular sections were washed and incubated for 2h at room temperature with biotinylated secondary antiserum (goat anti-rabbit IgG serum, 1:200 for immunodetection of Iba1 and 1:500 for immunodetection of COX2 and StAR from Vector Laboratories Inc., Burlingame, CA, USA) diluted in incubation buffer (2% goat normal serum in PBS for immunodetection of COX2 and StAR or 5% BSA 0.1% Triton prepared in PBS for immunodetection of Iba1).

Techniques: Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Alterations in oxidative, inflammatory and apoptotic events in short-lived and long-lived mice testes

doi:

Figure Lengend Snippet: Panel ( A ) COX2-immunoreactive interstitial cells were isolated by Laser Capture Microdissection (LCM) from testicular sections of short-lived (GH-Tg) and long-lived (Ames dwarf and GHRH-KO) mice. The same section is illustrated before (left panel) and after (right panel) LCM. Bar, 25 μm. A total of 50 to 80 COX2-immunopositive interstitial cells were isolated by LCM and subsequently used to evaluate the expression of CD68 (macrophage cell marker) and StAR (Leydig cell marker) by RT-PCR. Panels ( B and C ) Immuno-colocalization of COX2 and Iba1 (macrophage cell marker; Panel ( B ) and immuno-colocalization of COX2 and StAR (Leydig cell marker; Panel ( C ) in testicular sections from a short-lived mouse (GH-Tg) was examined using a light microscope. Bar, 20 (m. Similar images were seen when testicular sections from long-lived (Ames dwarf and GHRH-KO) mice were used (data not shown).

Article Snippet: Testicular sections were washed and incubated for 2h at room temperature with biotinylated secondary antiserum (goat anti-rabbit IgG serum, 1:200 for immunodetection of Iba1 and 1:500 for immunodetection of COX2 and StAR from Vector Laboratories Inc., Burlingame, CA, USA) diluted in incubation buffer (2% goat normal serum in PBS for immunodetection of COX2 and StAR or 5% BSA 0.1% Triton prepared in PBS for immunodetection of Iba1).

Techniques: Isolation, Laser Capture Microdissection, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Light Microscopy

Journal: Aging (Albany NY)

Article Title: Alterations in oxidative, inflammatory and apoptotic events in short-lived and long-lived mice testes

doi:

Figure Lengend Snippet: Panel ( A ) the relative expression levels of CD68 and CD163 (macrophage cell markers) and the relative expression levels of StAR (Leydig cell marker) were determined by real time-PCR in testicular homogenates of short-lived (GH-Tg) and long-lived (GHRH-KO and Ames dwarf) mice and their normal littermates. Results were normalized to GAPDH housekeeping gene and expressed as fold change relative to the control (normal littermates), which was assigned a value of 1. Bar plot graphs represent the mean + SEM; n = 5-7. * p < 0.05, t-Student test. Panels ( B and C ) Quantification of Iba1-positive macrophages in testes of GH-Tg short-lived mice, GHRH-KO and Ames dwarf long-lived mice and their normal littermates was evaluated using a light microscope with a magnification of 400x and a gridded eyepiece. Results are expressed as macrophages/mm 2 (Panel B ) and macrophages/tubule (Panel C ). Bar plot graphs represent the mean + SEM; n = 5-7. * p < 0.05, t-Student test.

Article Snippet: Testicular sections were washed and incubated for 2h at room temperature with biotinylated secondary antiserum (goat anti-rabbit IgG serum, 1:200 for immunodetection of Iba1 and 1:500 for immunodetection of COX2 and StAR from Vector Laboratories Inc., Burlingame, CA, USA) diluted in incubation buffer (2% goat normal serum in PBS for immunodetection of COX2 and StAR or 5% BSA 0.1% Triton prepared in PBS for immunodetection of Iba1).

Techniques: Expressing, Marker, Real-time Polymerase Chain Reaction, Light Microscopy