Journal: Iranian Journal of Basic Medical Sciences

Article Title: Tempol relieves lung injury in a rat model of chronic intermittent hypoxia via suppression of inflammation and oxidative stress

doi: 10.22038/ijbms.2018.31716.7714

Figure Lengend Snippet: Tempol further promoted intermittent hypoxia-induced activation of the Nrf2/HO-1 signaling pathway. (A) The level of HO-1 in lung tissues was detected by a commercial kit. (B) The protein levels of HO-1, cytoplasmic Nrf2, and nuclear Nrf2 in lung tissues were measured by Western blot assay. GAPDH was used as a loading control. (C)& (D) The gray-scale value of the bands was quantitatively analyzed. (E) DNA binding activity of Nrf2 in lung tissues was determined by EMSA. (F) Quantitative analysis of Nrf2 DNA binding activity. The experimental data were expressed as mean±SD (n=6). * P< 0.05, *** P< 0.001, versus the NA group. ### P< 0.001, versus the IH group. ns, no significance, versus the NA group

Article Snippet: Whereafter the membranes were blocked with 5% skimmed milk for 1 hr and incubated with corresponding primary antibodies against COX2 (1:700, Proteintech, USA), iNOS (1:7500, Proteintech, USA), IκBα (1:1000, Cell Signaling Technology, USA), p- IκBα (1:1000, Cell Signaling Technology, USA), NF-κB (1:1000, Proteintech, USA), HO-1 (1:500, Boster, China), Nrf2 (1:500 Boster, China), GAPDH (1:5000, Bioss, China), and Histone H3 (1:1000, Proteintech, USA) at 4 ° C overnight.

Techniques: Activation Assay, Western Blot, Control, Binding Assay, Activity Assay

Journal: PLoS ONE

Article Title: Alpha-Synuclein Proteins Promote Pro-Inflammatory Cascades in Microglia: Stronger Effects of the A53T Mutant

doi: 10.1371/journal.pone.0162717

Figure Lengend Snippet: Nuclear localization of the p65 subunit, c-Fos (subunit of AP-1) as well as Nrf2 was evaluated on microglial cells after a 5 μM A53T exposure for 30 min, 1 h or 2 h (Fig 7A). These chemiluminescent detection assays were realized by western-blot with 10 μg of nuclear proteins. The histone deacetylase 1 (HDAC1) was used as a loading control. Furthermore, p65, c-Fos and Nrf2 detected proteins were normalized and quantified (Fig 7B). Results are given as mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, significantly different from control condition. A cytoplasmic protein negative control western-blot (αTub) was also realized (Fig 7C) to demonstrate the effectiveness of the nuclear isolation.

Article Snippet: Blots were incubated with a mouse anti-Stat1 (1:500, BD Biosciences, Belgium), rabbit anti-phospho-Stat1 (1:1000, Bioke, The Netherlands), rabbit anti-p38 (1:1000, Calbiochem, Belgium), rabbit anti-phospho-p38 (1:1000, Bioke), rabbit anti-ERK (1:20000, Sigma), rabbit anti-phospho-ERK (1:2000, Bioke), mouse anti-α-tubulin (1:5000, AbCam, UK), rabbit anti-p65 (1:200, Santa Cruz Biotechnologies, Germany), rabbit anti-c-Fos (1:1000, Bioke), rabbit anti-Nrf2 (1:1000, Bioke) and mouse anti-HDAC1 (1:2000, AbCam) antibody.

Techniques: Western Blot, Histone Deacetylase Assay, Negative Control, Isolation

Journal: Journal of Animal Science and Biotechnology

Article Title: Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

doi: 10.1186/s40104-021-00595-x

Figure Lengend Snippet: Effects of PQQ on proteins expression of Nrf2 and HO-1 in cells cultured with H 2 O 2 . a , Immunofluorescence staining of cytoplasmic and nuclear Nrf2 in IPEC-J2 cells. The scale bar represents 50 μm. b , Western blot analysis of nuclear Nrf2, cytoplasmic Nrf2, and HO-1. Densitometric values were normalized to those of PCNA or β-actin, as appropriate; c-e , Statistical analysis of the data in B. CTRL, cells cultured with basal medium; H 2 O 2 , cells cultured with 200 μmol/L H 2 O 2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both 200 μmol/L H 2 O 2 and 10 nmol/L PQQ for another 2 h. n = 3

Article Snippet: In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan).

Techniques: Expressing, Cell Culture, Immunofluorescence, Staining, Western Blot, Incubation

Journal: Journal of Animal Science and Biotechnology

Article Title: Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

doi: 10.1186/s40104-021-00595-x

Figure Lengend Snippet: Effects of PQQ on levels of Nrf2 and HO-1 in cells treated with siRNA. a Nrf2 knockdown efficiency in cells transfected with three different sequences of Nrf2 siRNA or with NC siRNA. b and c mRNA expression levels of Nrf2 and HO-1 in IPEC-J2 cells transfected with or without Nrf2 siRNA-2. d Western blot analysis of Nrf2 and HO-1 in cells transfected with or without Nrf2 siRNA-2. e Statistical analysis of the data in D. CTRL, cells cultured with basal medium; H 2 O 2 , cells cultured with 200 μmol/L H 2 O 2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H 2 O 2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H 2 O 2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H 2 O 2 for another 2 h. * denotes a significant difference ( P < 0.05) with respect to the CTRL group. # denotes a significant difference ( P < 0.05) with respect to the H 2 O 2 group. n = 3

Article Snippet: In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Cell Culture, Incubation

Journal: Journal of Animal Science and Biotechnology

Article Title: Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

doi: 10.1186/s40104-021-00595-x

Figure Lengend Snippet: Effects of PQQ on levels of ROS and ROS-regulated proteins in cells treated with siRNA. a Level of ROS in IPEC-J2 cells treated with or without Nrf2 siRNA-2. b-c Ratio of Bcl-2/Bax mRNA levels and mRNA expression levels of Caspase-3 in IPEC-J2 cells treated with or without Nrf2 siRNA-2. d Western blot analysis of cytoplasmic Bax, Bcl-2 and Caspase-3. Densitometric values were normalized to those of β-actin. e Statistical analysis of the data in D. f Statistical analysis of the Bcl-2/Bax protein ratio. CTRL, cells cultured with basal medium; H 2 O 2 , cells cultured with 200 μmol/L H 2 O 2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H 2 O 2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H 2 O 2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H 2 O 2 for another 2 h. n = 3

Article Snippet: In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan).

Techniques: Expressing, Western Blot, Cell Culture, Incubation, Transfection

Journal: Journal of Animal Science and Biotechnology

Article Title: Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

doi: 10.1186/s40104-021-00595-x

Figure Lengend Snippet: Effects of PQQ on tight junctions expression and cell viability in cells treated with siRNA. a Western blot analysis of ZO-1, ZO-2, Occludin and Claudin-1 in IPEC-J2 cells treated with or without Nrf2 siRNA-2. The densitometric values were normalized to those of β-actin. n = 3. b Viability of IPEC-J2 cells treated with or without Nrf2 siRNA-2 and incubated in the presence or absence of H 2 O 2 or PQQ. CTRL, cells cultured with basal medium; H 2 O 2 , cells cultured with 200 μmol/L H 2 O 2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H 2 O 2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H 2 O 2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H 2 O 2 for another 2 h. n = 6. * denotes a significant difference ( P < 0.05) with respect to the CTRL group. # denotes a significant difference ( P < 0.05) with respect to the H 2 O 2 group

Article Snippet: In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan).

Techniques: Expressing, Western Blot, Incubation, Cell Culture, Transfection