mouse primary ab against β actin  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: Adipocyte STAT5 deficiency does not affect blood glucose homeostasis in obese mice

doi: 10.1371/journal.pone.0260501

Figure Lengend Snippet: Abundance of total STAT3 and of phosphorylated STAT3 (pSTAT3) normalised to β-actin in epidydimal fat (A) and UCP1 expression normalised to β-actin in the inguinal fat (B) from 23–24 weeks old male mice fed high-fat diet ad libitum which lack STAT5 in their adipocytes ( Stat5 Adipoq / KO) as compared to their wild type controls (WT). No significant differences WT vs Stat5 Adipoq .

Article Snippet: For the detection of STAT3 and phospho-STAT3 Rabbit primary Ab against STAT3 and phospho-STAT3 Tyr705 (#4904 and #9145, Cell Signaling Technology, Danvers, MA, USA) were used at a dilution of 1:1,000 in TBST, and Mouse primary Ab against β-actin as house-keeping protein (#ab8226, Abcam, Cambridge, UK) were used at a dilution of 1:5,000 in TBST.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Adipocyte STAT5 deficiency does not affect blood glucose homeostasis in obese mice

doi: 10.1371/journal.pone.0260501

Figure Lengend Snippet: Abundance of total STAT3 and of phosphorylated STAT3 (pSTAT3) normalised to β-actin in epidydimal fat (A) and UCP1 expression normalised to β-actin in the inguinal fat (B) from 23–24 weeks old male mice fed high-fat diet ad libitum which lack STAT5 in their adipocytes ( Stat5 Adipoq / KO) as compared to their wild type controls (WT). No significant differences WT vs Stat5 Adipoq .

Article Snippet: For the detection of STAT3 and phospho-STAT3 Rabbit primary Ab against STAT3 and phospho-STAT3 Tyr705 (#4904 and #9145, Cell Signaling Technology, Danvers, MA, USA) were used at a dilution of 1:1,000 in TBST, and Mouse primary Ab against β-actin as house-keeping protein (#ab8226, Abcam, Cambridge, UK) were used at a dilution of 1:5,000 in TBST.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Bacteroides fragilis Enterotoxin Induces Sulfiredoxin-1 Expression in Intestinal Epithelial Cell Lines Through a Mitogen-Activated Protein Kinases- and Nrf2-Dependent Pathway, Leading to the Suppression of Apoptosis

doi: 10.3390/ijms21155383

Figure Lengend Snippet: Effects of Nrf2-inhibition on Srx-1-expression in BFT-exposed HCT 116 cells. ( A ) BFT (300 ng/mL) was added to HCT 116 cells for the indicated periods of time. Nuclear protein expression of phospho-Nrf2 and lamin B was examined by western blot. Positive controls (+) were obtained from cells that were treated with 20 µM of sulforaphane for 2 h. Bottom panel shows densitometric analysis for expressed proteins. Values represent the relative densities of each protein compared with lamin B (mean ± SD, n = 3); ( B ) BFT (300 ng/mL) was added to HCT 116 cells for the indicated periods. Nrf2 DNA binding activity was assessed by EMSA; ( C ) HCT 116 cells were treated with BFT (300 ng/mL) for 6 h and nuclear extracts were obtained. Supershift assays using nuclear extracts were performed using anti-Nrf2 Ab and IgG isotype control Ab; ( D ) HCT 116 cells were treated with BFT (300 ng/mL) for 6 h. Nuclear extracts were added a 100-fold excess of the unlabeled probe (cold probe) or a mutant probe to the reaction, after which radiolabeled probes were added. After then, EMSA was performed identical to ( B ); ( E ) HCT 116 cells were transfected with shRNA against Nrf2 or control RNA. BFT (300 ng/mL) was added to each group for 6 h (phospho-Nrf2, top panels) or 18 h (Srx-1, bottom panels). Nuclear protein expression of phospho-Nrf2 and lamin B was determined by western blot. Concurrently, protein-expression of Srx-1 and actin was also evaluated by western blot; ( F ) HCT 116 cells were transfected with siRNA against Nrf2 or NS-RNA. BFT (300 ng/mL) was added to each group for 6 h (phospho- Nrf2, top panels) or 18 h (Srx-1, bottom panels). Analysis of protein-expression was identical to ( E ). All images are representative of more than three independent experiments.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) supported mouse primary Abs against actin (sc-47778) and lamin B (sc-374,015) and goat anti-mouse (sc-2005) and anti-rabbit (sc-2004) Abs conjugated to horseradish peroxidase.

Techniques: Inhibition, Expressing, Western Blot, Binding Assay, Activity Assay, Control, Mutagenesis, Transfection, shRNA

Journal: Cancer Science

Article Title: p62 promotes bladder cancer cell growth by activating KEAP1/NRF2‐dependent antioxidative response

doi: 10.1111/cas.14321

Figure Lengend Snippet: p62 promotes bladder cancer cell proliferation and inhibits its apoptosis in vitro. A‐D, MTT cell proliferation and colony formation assay after p62 knockdown and overexpression. E, F, Immunoblots of B‐cell lymphoma 2 (Bcl‐2) and Bcl‐2‐associated X protein (Bax) after p62 knockdown and overexpression. Data are shown as the mean ± SD, Student’s t test, * P < .05, ** P < .01. Con, control; sg, single guide; Vec, vector

Article Snippet: Mouse primary Abs against Bcl‐2 and β‐actin were purchased from Cell Signaling Technology.

Techniques: In Vitro, MTT Cell Proliferation, Colony Assay, Over Expression, Western Blot, Plasmid Preparation

Journal: Cancer Science

Article Title: p62 promotes bladder cancer cell growth by activating KEAP1/NRF2‐dependent antioxidative response

doi: 10.1111/cas.14321

Figure Lengend Snippet: Knockdown of p62 inhibits bladder cancer growth in vivo. A, 5637 cells were infected with single guide (sg) p62 and cultured for 48 h and then inoculated into nude mice. Tumor volume was calculated at indicated time points. B, C, Tumor appearance and weight of control and p62‐depleted 5637 cells are shown. D, Immunoblots of indicated proteins of tumors from control and p62‐depleted 5637 cell‐induced tumor. E, Representative immunohistochemistry images of nuclear factor‐E2‐related factor 2 (NRF2) and glutamate‐cysteine ligase catalytic subunit (GCLC). Scale bar, 50 μm. Data are shown as the mean ± SD, Student’s t test, * P < .05, ** P < .01. Bcl‐2, B‐cell lymphoma‐2; Con, control; KEAP1, Kelch‐like ECH‐associated protein 1

Article Snippet: Mouse primary Abs against Bcl‐2 and β‐actin were purchased from Cell Signaling Technology.

Techniques: In Vivo, Infection, Cell Culture, Western Blot, Immunohistochemistry