Journal: International Journal of Molecular Sciences

Article Title: In Vitro Tumor Cell-Binding Assay to Select High-Binding Antibody and Predict Therapy Response for Personalized 64 Cu-Intraperitoneal Radioimmunotherapy against Peritoneal Dissemination of Pancreatic Cancer: A Feasibility Study

doi: 10.3390/ijms23105807

Figure Lengend Snippet: Relationships between Western blots for EGFR expression and in vitro cell-binding assay with 64 Cu-anti-EGFR antibodies (cetuximab). ( A ) Representative images of Western blots for EGFR and GAPDH expression. ( B ) Correlation between relative EGFR expression (EGFR/GAPDH) from Western blots and cell binding (%) from an in vitro cell-binding assay with 64 Cu-anti-EGFR antibodies (cetuximab). ( C ) Coefficient of variation (%) for relative EGFR expression from Western blots (Western blots) and cell binding (%) from an in vitro cell-binding assay (cell binding).

Article Snippet: For the primary antibodies, rabbit anti-EGFR antibodies (4267, Cell Signaling Technology) and mouse anti-GAPDH antibodies (MCA4739, AbD Serotec, Oxford, UK) were used as loading controls.

Techniques: Western Blot, Expressing, In Vitro, Cell Binding Assay, Binding Assay

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression inhibits HuT78 cell growth and proliferation. HuT78 cells expressing hemagglutinin (HA)-tagged SAMHD1 were generated via lentiviral vector-mediated transduction. (A) Exogenous expression of SAMHD1 was validated by immunoblotting analysis using antibodies to HA (SAMHD1) and GAPDH (loading control). (B) Total RNA was isolated from HuT78 vector or SAMHD1-expressing cells and qPCR analysis was performed to quantify the relative SAMHD1 mRNA levels. GAPDH mRNA levels were quantified as internal control. (C) HuT78 vector control and SAMHD1 expressing cells were seeded in triplicate in 24-well plates at a density of 1 × 104 cells/well in 1 ml culture media on day 0 and live cells were counted on indicated days via trypan-blue exclusion method. (D) Cell lines were seeded on day 0 in 4 replicates in 96-well plates at density of 1 × 103 cells/well in 100 µl and cell proliferation was determined on indicated days utilizing an MTS-based proliferation assay. All the data presented are representative of 3 or more independent experiments. (C-D) ***, p < 0.0001.

Article Snippet: 65 Immunoblotting was performed using the following antibodies: HA-11 (1:1000, Covance #901501), caspase-8 (1:1000, Cell signaling #9746S), caspase-3 (1:1000, Cell signaling #9662S), PARP (1:1000, Cell Signaling, #9542S), FLIP S/L (1:500, Santa Cruz Biotechnology sc-5276) and GAPDH (1:3000, Bio-Rad #AHP1628).

Techniques: Expressing, Generated, Plasmid Preparation, Transduction, Western Blot, Control, Isolation, Proliferation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of matrix metalloproteinase expression by selective clearing of senescent dermal fibroblasts attenuates ultraviolet-induced photoaging.

doi: 10.1016/j.biopha.2022.113034

Figure Lengend Snippet: Fig. 2. Selective elimination of senescent cells in UV-irradiated mice skin after treatment with ABT-263 or ABT-737. Hairless mice were exposed to chronic UV irradiation for eight weeks with 100 mJ/cm2 for the first 2 weeks, then 50 mJ/cm2 weekly, then 300 mJ/cm2. Each drug (ABT-263, 5 μM; ABT-737, 10 μM) or vehicle (DMSO) was diluted in PBS (30 μl) and injected intradermally into the dorsal skin for a total of ten doses. (A) Representative images of SA-β-gal staining in mice skin. SA-β-gal (blue color) was detected in 6 µm frozen skin sections from unirradiated control mice injected with vehicle (n = 7, white bars), UVB-irradiated mice injected with vehicle (n = 4, black bars), UVB-irradiated mice injected with ABT-263 (n = 6, blue bars) or ABT-737 (n = 7, red bars). Arrowheads indicate stained cells. (B) Quantitative analysis of the percentage of SA-β-gal positive cells in the indicated mice skin (n = 3–4 mice per group) (C) Western blots and quantification of p21 and p16 protein levels normalized to GAPDH using protein extracts from the skin. (D) p16 and p21 mRNA levels in indicated mice skin, standardized to GAPDH. (E) Representative images of immunofluorescence staining for p16 (red) and DAPI (blue) in the skin of the indicated mice. Right: Quan tification of the percentage of p16-positive cells among DAPI-positive cells in over n = 4 mice per group. Arrowheads indicate stained cells. Data represent means ± SEM, *p < 0.05, * *p < 0.005, * **p < 0.001. p-values were calculated by Student’s t-test. A, Bar = 100 µm; E, Bar = 50 µm.

Article Snippet: The antibodies used included p16 (MA5–17142, Thermo Fisher Scientific; ab189034, Abcam), p21 (556431, BD Pharmingen; ab109199, Abcam), GAPDH (CSB-PA00029A0Rb, Cusabio, Houston, TX, USA), β-actin (CSBPA000350, Cusabio), MMP-1 (Lab Frontier, Seoul, Korea), and type I procollagen (SP1.

Techniques: Irradiation, Injection, Staining, Control, Western Blot, Immunofluorescence

Journal: iScience

Article Title: O 6 -methylguanine DNA methyltransferase regulates β-glucan-induced trained immunity of macrophages via farnesoid X receptor and AMPK

doi: 10.1016/j.isci.2023.108733

Figure Lengend Snippet:

Article Snippet: The antibodies were diluted in the buffer according to the the manufacturer’s protocol at the following concentration: rat anti-mMGMT, 1:2000 (R&D Systems, USA); rabbit anti-phospho AKT (Thr308), 1:1000; rabbit anti-AKT, 1:1000; rabbit anti-phospho NF-κB p65, 1:2000; rabbit anti-NF-κB p65, 1:4000; rabbit anti-phospho ERK1/2, 1:2000; rabbit anti-ERK1/2, 1:4000; rabbit anti-phospho p38, 1:2000; rabbit anti-p38, 1:4000; rabbit anti-phospho SAPK-JNK, 1:2000; rabbit anti-SAPK-JNK, 1:4000; rabbit anti-phospho mTOR, 1:1000; rabbit anti-mTOR, 1:1000; rabbit anti-HIF1α, rabbit anti-phospho AMPK alpha, 1:2000; rabbit anti-AMPK alpha, 1:2000; anti-actin antibody, 1:10000; and goat anti-rabbit IgG HRP, 1:4000 (all antibodies were purchased from Cell Signaling Technology, USA); goat anti-rat IgG HRP, 1:4000 (Biolegend, USA); sheep anti-mouse IgG HRP, 1:4000 (Cytiva, USA); rabbit polyclonal anti-GAPDH, 1:4000 (Bio-Rad, USA).The signal was detected by the ECL chemiluminescent detection method.

Techniques: Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Western Blot, Control, Software, SYBR Green Assay