Journal: PLoS ONE

Article Title: Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

doi: 10.1371/journal.pone.0012520

Figure Lengend Snippet: K562 cells with or without ME2 knockdown were lysed with RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA) containing 1 mM PMSF and a protease inhibitor cocktail and subjected to centrifugation at 15,000× g for 10 min at 4°C to remove debris. After lysis, equal aliquots of protein lysate were resolved by Western blotting. Western blots were probed with anti-phospho-ERK1/2, anti-ERK1, anti-p-AKT308, anti-AKT472, anti-AKT1/2, anti-GATA-1, anti-vimentin and anti-β-tubulin. A, phospho-ERK1/2 activity in ME2 knockdown K562 cells. B, Phospho-AKT detection in ME2 knockdown K562 cells. C, 10 µM PI3K inhibitor, LY294002, inhibits p-AKT activity. D, LY294002 rescue of differentiation in ME2 knockdown K562 cells. E, The effect of LY294002 on the proliferation of K562 cells with or without ME2 knockdown. F, The expression difference of GATA-1 and vimentin in ME2 knockdown cells. Data are representative of three independent experiments.

Article Snippet: The anti-GATA-1, phospho-ERK1/2, phospho-AKT308, phospho-AKT473 and AKT1/2 polyclonal antibodies were purchased from Cell Signaling Technology.

Techniques: Lysis, Protease Inhibitor, Centrifugation, Western Blot, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Adiponectin Agonist ADP355 Attenuates CCl 4 -Induced Liver Fibrosis in Mice

doi: 10.1371/journal.pone.0110405

Figure Lengend Snippet: A – B ) Western blot analysis showed that CCl 4 reduced the phosphorylation of eNOS and AMPK, while ADP355 treatment significantly increased the phosphorylation of eNOS ser1177 and AMPK tyr172 residue. C ) Western blot and densitometric ratio of p-AKT ser473/AKT, * p <0.05 (N = 5 mice/cohort).

Article Snippet: The primary antibodies were purchased from following manufacturer; CTGF, TGF-β1and MMP13 (Santa Cruz Biotechnology, Dallas, Texas); p-AMPKα tyr172, AMPKα, p-eNOS ser1177, eNOS, pAKT ser473 and AKT (Cell Signaling, Beverly, MA); anti-actin, α-SMA and anti-desmin (Sigma-Aldrich, St. Louis, MO); anti-mTIMP1 (R&D Systems, Minneapolis, MN).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

doi: 10.1371/journal.pone.0004077

Figure Lengend Snippet: (A) The rMC-1 cells were treated with 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca 2+ ] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H 2 O 2 -induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H 2 O 2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H 2 O 2 . The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H 2 O 2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P<0.01, under H 2 O 2 exposure from 1.5 h to 4 h; the U73122 and SERPINA3K-treated cells versus the BSA-treated cells. * P<0.01, versus control cells. # P<0.01, the cells treated with H 2 O 2 , m-3M3FBS and SERPINA3K versus the cells treated with H 2 O 2 and SERPINA3K. & P<0.01, versus control cells exposed to H 2 O 2 for 0 min.

Article Snippet: Antibodies for PLCγ1 (t-PLC), phosphorylated PLCγ1 at Tyr783 (p-PLC), ERK1/2 (t-ERK), phosphorylated ERK1/2 at Thr202 and Tyr204 (p-ERK), Akt (t-AKT) and phosphorylated Akt at Ser473 (p-AKT) were purchased from Cell Signaling (Danvers, MA) and used at the 1∶1000 dilution.

Techniques: Negative Control, Fluorescence, MTT Assay, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models

doi: 10.1016/j.jcmgh.2021.11.007

Figure Lengend Snippet: List of Antibodies and Relative Dilutions Used in Western Blot and Immunocytochemistry Experiments

Article Snippet: Anti–P-Ser473 Akt, Akt, P(Thr37/46)-4E-BP1, 4E-BP1, P(Ser2448)-mTOR, and mTOR antibodies were acquired from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Immunocytochemistry