For flow cytometry analyses, Neuro2a cells were seeded in 2 × 106cells/well density in 6-well plate and incubated for 12 h at 37°C, 5% CO2 incubator. JEV infection were given in three wells at 0.1MOI. The 1b and 1f treatment was given at 15μM concentration separately in JEV infected and non-infected cells 2 h post-infection. Untreated and uninfected controls were kept along with one DMSO (0.1%) control. After 48 h of incubation, cells were pelleted and washed with 1% phosphate buffer saline (PBS, pH 7.4). Further, cells were suspended in 500μl of 1% PBS and incubated with 10μM 2′,7′-dichlorodihydrofluorescin diacetate (H2DCF-DA) for 30 min at 37°C. The stained cells were analyzed using flow cytometry (BC, CytoFlex S Analyser). The DJ-1(5933, CST, USA), SQSTM1 (39749, CST, USA), HO-1 (43966, CST, USA), NQO1 (62262, CST, USA), antibodies were used to explore the modulation of NRF2-SQSTM1 pathway in the cells. The β-actin was used as a loading control for all the experiments (MA1-91399, Invitrogen).
Ma191399
Manufactured by Thermo Fisher Scientific
The MA191399 is a laboratory equipment product offered by Thermo Fisher Scientific. It serves as a core function within the laboratory setting, though the specific intended use is not provided in this factual, unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
60 mm culture plate, by Tarsons (2 mentions)
Ma5 15073, by Thermo Fisher Scientific (2 mentions)
Ahf0082, by Thermo Fisher Scientific (2 mentions)
Ab209780, by Abcam (2 mentions)
Irdye 800cw donkey anti rabbit, by LI COR (2 mentions)
Pa1 26204, by Thermo Fisher Scientific (2 mentions)
Ma5 15122, by Thermo Fisher Scientific (2 mentions)
Irdye 680rd donkey anti mouse secondary antibodies, by LI COR (2 mentions)
Goat anti rabbit 680rd, by LI COR (1 mentions)
Goat anti mouse 800cw, by LI COR (1 mentions)
Sapphire biomolecular imager, by Azure Biosystems (1 mentions)
6 protocols using ma191399
1
Neuro2a Cells JEV Infection Analysis
2
TLR3-Induced Signaling Pathway Analysis
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Cells were cultured at a density of 100 × 104 cells in a 60-mm culture plate (Tarsons-960020). Four hours before the addition of the TLR3 ligand, the MyD88 inhibitor was added and incubated for 24 h. Cells were lysed using RIPA buffer, and gel electrophoresis was performed using acrylamide gel. Proteins were transferred to PVDF membranes and blotted with antibodies against interleukin 1 receptor-associated kinase 1 (IRAK1; Invitrogen-38-5600), phospho IRAK1–Thr209, (Invitrogen-PA5-38633), transforming growth factor beta-activated kinase 1 (TAK1; Invitrogen-700 113), phospho TAK1–Thr184/187 (Invitrogen-MA5-15073), TGF-beta-activated kinase 1 (TAB1; Invitrogen-PA5-28683), TNF receptor-associated factor 6 (TRAF6; Invitrogen-PA5-29622), and cyclin D1 (Invitrogen-AHF0082). The antibody against β-actin (Invitrogen-MA191399) was used as a loading control.
3
TLR3 Signaling Pathway Regulation
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Cells were cultured at a density of 100x10 4 cells in 60 mm culture plate (Tarsons-960020). Four hours before the addition of TLR3 ligand, MyD88 inhibitor was added and incubated for 24 hours. Cells were lysed using RIPA buffer and gel electrophoresis was performed using acrylamide gel. Proteins were transferred to PVDF Membranes and blotted with antibodies against IRAK1 (Invitrogen-38-5600), phospho IRAK1-Thr209, (Invitrogen-PA5-38633), TAK1 (Invitrogen-700 113 ), phospho TAK1-Thr184/187 (Invitrogen-MA5-15073), TAB1
(Invitrogen-PA5-28683), TRAF-6 (Invitrogen-PA5-29622), and Cyclin D1 (Invitrogen-AHF0082). The antibody against β-actin (Invitrogen-MA191399) was used as a loading control.
(Invitrogen-PA5-28683), TRAF-6 (Invitrogen-PA5-29622), and Cyclin D1 (Invitrogen-AHF0082). The antibody against β-actin (Invitrogen-MA191399) was used as a loading control.
4
Western Blot Analysis of PIP5K1γ Expression
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Protein from cultured fibroblasts was harvested and lysates were blotted as previously described.111 (link) anti-PIP5K1γ (ABS190; 1:300; Sigma) and anti-β-actin (MA1-91399; 1:1000; Thermo-Fisher) were applied to transferred membranes overnight at 4°C. Blot bands were detected by Sapphire Biomolecular Imager (Azure Biosystems) after 1 h incubation in the following secondary antibodies: goat anti-rabbit 680RD (P/N 926–68071, 1:10,000; LI-COR), goat anti-Mouse 800CW (P/N 925–32210, 1:10,000; LI-COR). Images were processed on ImageJ using the BioImporter plugin tool to calculate the protein expression for each band. Protein abundance was first normalized to beta-actin intensity then normalized to control cell intensity.
5
Immunofluorescence analysis of ECM proteins
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Primary antibodies used in this study were: recombinant rabbit anti-cellular communication network factor 2 (CCN2) monoclonal antibody diluted to a concentration of 1:1,000 (Abcam, ab209780); rabbit anti-bone morphogenetic protein 1 (BMP1) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; PA5-103,660; RRID: AB_2852994); rabbit anti-collagen type I (COL1) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; PA1-26204; RRID: AB_2260734); rabbit anti-suppressors of mothers against decapentaplegic homolog 2 (SMAD2) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; 51–1300; RRID: AB_2533896); rabbit anti-phosphorylated SMAD2 (pSMAD2) monoclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; MA5-15122; RRID: AB_10978317); and mouse anti-beta actin (β-actin) monoclonal antibody diluted to a concentration of 1:5,000 (Thermo Fisher Scientific; MA1-91399; RRID: AB_2273656).
For near-infrared fluorescence detection, IRDye® 800CW donkey anti-rabbit and IRDye® 680RD donkey anti-mouse secondary antibodies (LI-COR, 926-32213 and 926-68072, respectively) were used.
For near-infrared fluorescence detection, IRDye® 800CW donkey anti-rabbit and IRDye® 680RD donkey anti-mouse secondary antibodies (LI-COR, 926-32213 and 926-68072, respectively) were used.
6
Quantification of Extracellular Matrix Proteins
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Primary antibodies used in this study were: recombinant rabbit anti-cellular communication network factor 2 (CCN2) monoclonal antibody diluted to a concentration of 1:1,000 (Abcam, ab209780); rabbit anti-bone morphogenetic protein 1 (BMP1) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; PA5-103660; RRID: AB_2852994); rabbit anti-collagen type I (COL1) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; PA1-26204; RRID: AB_2260734); rabbit anti-suppressors of mothers against decapentaplegic homolog 2 (SMAD2) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; 51-1300; RRID: AB_2533896); rabbit anti-phosphorylated SMAD2 (pSMAD2) monoclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; MA5-15122; RRID: AB_10978317); and mouse anti-beta actin (β-actin) monoclonal antibody diluted to a concentration of 1:5,000 (Thermo Fisher Scientific; MA1-91399; RRID: AB_2273656).
For near-infrared fluorescence detection, IRDye® 800CW donkey anti-rabbit and IRDye® 680RD donkey anti-mouse secondary antibodies (LI-COR, 926-32213 and 926-68072, respectively) were used.
For near-infrared fluorescence detection, IRDye® 800CW donkey anti-rabbit and IRDye® 680RD donkey anti-mouse secondary antibodies (LI-COR, 926-32213 and 926-68072, respectively) were used.
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