Journal: Egyptian Journal of Medical Human Genetics

Article Title: Ankaferd Blood Stopper induces apoptosis and regulates PAR1 and EPCR expression in human leukemia cells

doi: 10.1016/j.ejmhg.2014.10.001

Figure Lengend Snippet: Figure 3 Apoptosis detection by Annexin V staining in K-562 and Jurkat cells after ABS treatment.

Article Snippet: Apoptosis detection was performed using Annexin V apoptosis detection kit (sc-4252 AK) (Santa Cruz Biotechnology, Inc.) on live cells according to the manufacturer’s recommendations.

Techniques: Staining

Journal: iScience

Article Title: ISG12a promotes immunotherapy of HBV-associated hepatocellular carcinoma through blocking TRIM21/AKT/β-Catenin/PD-L1 axis

doi: 10.1016/j.isci.2024.109533

Figure Lengend Snippet: Figure 1. Influence of HBV infection on malignant phenotypes of HLCZ01 cells (A) Immunoblots for p-STAT1, STAT1, p-STAT2, STAT2 levels in lysates of HLCZ01 cells with or without HBV infection. Cells were infected with NDV for 16 h, and the multiplicity of infection (MOI) of NDV was 0.2. ND, not detected. (B) Immunoblots for PD-L1, p-AKT, AKT, p-mTOR, and mTOR levels in lysates of HLCZ01 cells with or without HBV infection. (C) Flow cytometry analysis for surface PD-L1 level on HLCZ01 cells with or without HBV infection. (D) Proliferation of HLCZ01 cells with or without HBV infection. Cell proliferation was determined by counting using a blood counting chamber. (E) Immunofluorescence staining for F-actin level in HLCZ01 cells with or without HBV infection. Magnification: 6003, and scale bar: 20 mm. (F) Tumor formation of HLCZ01 cells with or without HBV infection in NCG mice (n = 5 or 6). Minimum scale of the ruler: 1 mm. Experiments were independently replicated at least two times with similar results. Data were analyzed by one-way ANOVA (A) or unpaired two-sided Student’s t tests (B–D and F), and are presented as mean G SEM with three replicate experiments (A and B) or at least three biological replicates (C, D, and F). **p < 0.01 and ***p < 0.001.

Article Snippet: Following antibodies were used for immunoblots and co-IP: ISG12a (1:1000, Cat# SAB1408588, Sigma-Aldrich), GAPDH (1:2000, clone 6C5, Cat#MAB374,MerckMillipore, Darmstadt, Germany), Flag (1:5000, clone M2, Cat# F3165, Sigma-Aldrich), V5 (1:2000, Cat# R960-25, Thermo Fisher Scientific), HA (1:1000, clone C29F4, Cat# 3724S, Cell Signaling Technology, Danvers, MA, USA), b-Catenin (1:1000, clone D10A8, Cat# 8480S, Cell Signaling Technology), p-b-Catenin (1:1000, clone D8E11, Cat# 5651S, Cell Signaling Technology), PD-L1 (1:250, Cat# 17952-1-AP, Proteintech, Rosemont, IL, USA), AKT (1:1000, clone C67E7, Cat# 4691S, Cell Signaling Technology), p-AKT (1:1000, clone D9E, Cat# 4060S, Cell Signaling Technology), mTOR (1:500, clone 30, Cat# sc-517464, Santa Cruz Technology, Dallas, TX, USA), p-mTOR (1:500, clone 59, Cat# sc-293133, Santa Cruz Technology), HBsAg (1:250, clone 1025, Cat# sc-53300, Santa Cruz Technology), HBcAg (1:250, clone C1-5, Cat# sc-23945, Santa Cruz Technology), b-actin (1:5000, cloneAC-74, Cat# A5316, Sigma-Aldrich), InVivoSIM anti-human PD-1 (NivolumabBiosimilar) (1:500, cloneNivolumab, Cat# SIM0003, Bio X Cell, NH, USA), goat anti-mouse IgG (HRP-linked) (1:5000, Cat# AP124P, Merck Millipore), goat anti-rabbit IgG (HRP-linked) (1:5000, 18 iScience 27, 109533, April 19, 2024 Cat# AP132P, Merck Millipore), and HRP-conjugated goat anti-human IgG (1:500, Cat# D110150, Sangon Biotech).

Techniques: Infection, Western Blot, Flow Cytometry, Staining

Journal: iScience

Article Title: ISG12a promotes immunotherapy of HBV-associated hepatocellular carcinoma through blocking TRIM21/AKT/β-Catenin/PD-L1 axis

doi: 10.1016/j.isci.2024.109533

Figure Lengend Snippet: Figure 2. Inhibitory effect of ISG12a on PD-L1 expression and cancer phenotypes of HBV-infected HLCZ01 cells (A) Immunoblots for ISG12a level in HLCZ01 cells with or without HBV infection. (B) Immunoblots for PD-L1, p-AKT, AKT, p-mTOR, mTOR, and ISG12a levels in lysates of HBV-infected HLCZ01 cells with ISG12a knockdown. (C) ELISA for HBsAg level in supernatants of HBV-infected HLCZ01 cells with ISG12a knockdown. (D) Flow cytometry analysis for surface PD-L1 level on HBV-infected HLCZ01 cells with ISG12a knockdown. (E) Proliferation of HBV-infected HLCZ01 cells with ISG12a knockdown. Cell proliferation was determined by counting using a blood counting chamber. (F) Immunofluorescence staining for F-actin level in HBV-infected HLCZ01 cells with ISG12a knockdown. Magnification: 6003, and scale bar: 20 mm. (G) Immunoblots for PD-L1, p-AKT, AKT, p-mTOR, mTOR, and Flag levels in HBV-infected HLCZ01 cells. The stably infected cells using lentivirus for silencing ISG12a expression were transfected with vector control or Flag-ISG12a plasmid for 72 h. (H) Immunofluorescence staining for F-actin level in HBV-infected HLCZ01 cells with the overexpression of ISG12a. Magnification: 6003, and scale bar: 20 mm.

Article Snippet: Following antibodies were used for immunoblots and co-IP: ISG12a (1:1000, Cat# SAB1408588, Sigma-Aldrich), GAPDH (1:2000, clone 6C5, Cat#MAB374,MerckMillipore, Darmstadt, Germany), Flag (1:5000, clone M2, Cat# F3165, Sigma-Aldrich), V5 (1:2000, Cat# R960-25, Thermo Fisher Scientific), HA (1:1000, clone C29F4, Cat# 3724S, Cell Signaling Technology, Danvers, MA, USA), b-Catenin (1:1000, clone D10A8, Cat# 8480S, Cell Signaling Technology), p-b-Catenin (1:1000, clone D8E11, Cat# 5651S, Cell Signaling Technology), PD-L1 (1:250, Cat# 17952-1-AP, Proteintech, Rosemont, IL, USA), AKT (1:1000, clone C67E7, Cat# 4691S, Cell Signaling Technology), p-AKT (1:1000, clone D9E, Cat# 4060S, Cell Signaling Technology), mTOR (1:500, clone 30, Cat# sc-517464, Santa Cruz Technology, Dallas, TX, USA), p-mTOR (1:500, clone 59, Cat# sc-293133, Santa Cruz Technology), HBsAg (1:250, clone 1025, Cat# sc-53300, Santa Cruz Technology), HBcAg (1:250, clone C1-5, Cat# sc-23945, Santa Cruz Technology), b-actin (1:5000, cloneAC-74, Cat# A5316, Sigma-Aldrich), InVivoSIM anti-human PD-1 (NivolumabBiosimilar) (1:500, cloneNivolumab, Cat# SIM0003, Bio X Cell, NH, USA), goat anti-mouse IgG (HRP-linked) (1:5000, Cat# AP124P, Merck Millipore), goat anti-rabbit IgG (HRP-linked) (1:5000, 18 iScience 27, 109533, April 19, 2024 Cat# AP132P, Merck Millipore), and HRP-conjugated goat anti-human IgG (1:500, Cat# D110150, Sangon Biotech).

Techniques: Expressing, Infection, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Stable Transfection, Transfection, Plasmid Preparation, Control, Over Expression

Journal: Journal of Biological Chemistry

Article Title: Inhibition of soluble epoxide hydrolase alleviates insulin resistance and hypertension via downregulation of SGLT2 in the mouse kidney

doi: 10.1016/j.jbc.2021.100667

Figure Lengend Snippet: Figure 2. HF–HS diet increased soluble epoxide hydrolase (sEH) and sodium–glucose cotransporter 2 (SGLT2) expression in the kidney. A, the expression of sEH and SGLT2 in mice kidneys was evaluated by immunohistochemical staining. The black arrow represents the high expression area of SGLT2. The scale bar represents 200 μm. B, quantitative analysis of immunohistochemical staining of sEH and SGLT2. C and D, representative Western blot and quantitation of sEH and SGLT2 expression. n = 6 mice in each group. Data are shown as mean ± SD. **p < 0.01. HF–HS, high-fat and high- salt; SC–NS, standard chow–normal salt.

Article Snippet: Antibodies against the following proteins were used in this study: sEH (sc-166961; dilution 1:500 for WB, 1:200 for IHC staining), SGLT2 (sc-393350; dilution 1:500 for WB, 1:200 for IHC), CD68 (SC-17832; dilution 1:1000 for WB, 1:500 for IHC), and tumor necrosis factor α (sc-12744; dilution 1:500 for WB, 1:250 for IHC) were purchased from Santa Cruz.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Inhibition of soluble epoxide hydrolase alleviates insulin resistance and hypertension via downregulation of SGLT2 in the mouse kidney

doi: 10.1016/j.jbc.2021.100667

Figure Lengend Snippet: Figure 4. TPPU administration lowered hypertension and improved insulin resistance by inhibiting sodium and glucose reabsorption mediated by 14,15-epoxyeicosatrienoic acid (14,15-EET)–triggered reduced sodium–glucose cotransporter 2 (SGLT2) expression. A, urine volume (n = 9 mice). B, urine sodium excretion (n = 6 mice). C, urine glucose excretion (n = 7 mice). D, representative immunohistochemical staining of SGLT2 in the kidney. The scale bar represents 100 μm. E and F, representative Western blot and quantitation of SGLT2 in mice kidney (n = 6 mice). G, soluble epoxide hydrolase (sEH) activity in kidney tissue (n = 4 mice). H, 14,15-EET levels in kidney tissue (n = 4 mice). Data are shown as mean ± SD. **p < 0.01. EET, epoxyeicosatrienoic acid; HF–HS, high-fat and high-salt; SC–NS, standard chow/normal salt; TPPU, trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea.

Article Snippet: Antibodies against the following proteins were used in this study: sEH (sc-166961; dilution 1:500 for WB, 1:200 for IHC staining), SGLT2 (sc-393350; dilution 1:500 for WB, 1:200 for IHC), CD68 (SC-17832; dilution 1:1000 for WB, 1:500 for IHC), and tumor necrosis factor α (sc-12744; dilution 1:500 for WB, 1:250 for IHC) were purchased from Santa Cruz.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitation Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Inhibition of soluble epoxide hydrolase alleviates insulin resistance and hypertension via downregulation of SGLT2 in the mouse kidney

doi: 10.1016/j.jbc.2021.100667

Figure Lengend Snippet: Figure 6. 14,15-Epoxyeicosatrienoic acid (14,15-EET) prevented the upregulation in sodium–glucose cotransporter 2 (SGLT2) expression in human proximal tubule epithelial cells (HK-2) treated with palmitic acid and NaCl via inhibiting the activation of the inhibitory kappa B kinase α/β (IKKα/ β)/NF-κB signaling pathway. A and B, human renal proximal tubule HK-2 cells were treated with 20 nM NaCl and different concentrations of palmitic acid, the Western blot and quantitation of SGLT2 and soluble epoxide hydrolase (sEH) expression. C, effects of trifluoromethoxyphenyl-3-(1-propionylpiperidin-4- yl) urea (TPPU) and four types of EETs on NaCl and palmitic acid induced elevated SGLT2 expression in HK-2 cells. D, effects of siEPHX2 on NaCl and palmitic acid induced elevated SGLT2 expression in HK-2 cells. E, effects of different concentrations of 14,15-EET on NaCl and palmitic acid induced activation of NF- κB pathway, bovine serum albumin (BSA) (solvent control), and mannitol (osmotic control). F, representative immunofluorescence staining of NF-κB (red) in

Article Snippet: Antibodies against the following proteins were used in this study: sEH (sc-166961; dilution 1:500 for WB, 1:200 for IHC staining), SGLT2 (sc-393350; dilution 1:500 for WB, 1:200 for IHC), CD68 (SC-17832; dilution 1:1000 for WB, 1:500 for IHC), and tumor necrosis factor α (sc-12744; dilution 1:500 for WB, 1:250 for IHC) were purchased from Santa Cruz.

Techniques: Expressing, Activation Assay, Western Blot, Quantitation Assay, Solvent, Control, Staining