Instantone elisa kit

Human IL10, IL6, and TNFα were analyzed using commercially available ELISA kits from Peprotech according to the manufacturer’s instructions. Fifty micrograms of brain extracts were analyzed in triplicate for each condition. Human p-STAT1 (Tyr701) and p-STAT3 (Tyr705) were analyzed using commercially available InstantOneELISA kit from eBioscience according to the manufacturer’s instructions. One hundred fifty micrograms of brain extracts was analyzed in duplicate for each condition. Descriptive statistics were calculated for every group and molecular subtype. Significances (p) were calculated with the SigmaStat Version 3.1 software (Systat Software Inc.) using the Student’s test/Mann–Whitney rank sum test, and for more than two groups Kruskal–Wallis test was used.

The RWP-1 pancreatic adenocarcinoma cell line was treated with BMS-754807 or OSI-906 for 1 or 6 h. Then, the culture medium was discarded, cells were washed with PBS and cell extracts were obtained to be assayed for phosphorylated MAPK levels using the InstantOne ELISA kit from eBioscience (San Diego, CA, USA), following the manufacturer’s instructions. Briefly, the cells were lysed in an appropriate volume of the cell lysis mix provided in the kit (100 µL for a 96-well plate) with shaking (300 rpm) at room temperature for 10 min. Then, 50 µL of cell lysate was added to each well of InstantOne ELISA microplates and, after the addition of 50 µL of the antibody cocktail, incubation was carried out for 1 h. Fifty µL of cell lysis mix or of the control lysate provided in the kit were used as negative and positive controls, respectively. Thereafter, three washes with 200 µL of wash buffer per well each time were carried out, and 100 µL of detection reagent was finally added. After shaking for 30 min, stop solution was added and the absorbance was read at 450 and 650 nm.

Samples of fluid leakage from mouse pleural cavities were adjusted using the Lowry method to contain the same protein concentrations (60 μg). The samples were transferred to a plate with microwells containing specific monoclonal antibodies against the phosphorylated protein p38 MAPK (phospho-p38 MAPK (Tyr180/Tyr182) Instant One ELISA kit, eBioscience, San Diego, California, USA\). Colorimetric measurements (450 nm) were performed on an ELISA plate reader, and the results are expressed as relative fold change when compared with the negative control group; this was used to represent the basal expression of phosphorylated p38 MAPK.

The InstantOne ELISA kit (eBioscience, San Diego, CA, USA) was used to measure levels of phosphorylated SMAD1 (Ser463/465) in whole embryo lysates. This kit is also predicted to detect the analogous phosphorylation sites of SMAD5 and SMAD8. Using a modified protocol, six organisms per well were lysed at 4 dpf with a combination of cell lysis buffer and mechanical homogenization. Lysate and phospho-SMAD1 (Ser463/465) capture and detection antibody reagents were added simultaneously to the InstantOne assay plate. After 1 hour of incubation, wells were washed and a detection solution was applied. Absorbance was measured at 450 nm. Positive control cell lysate and negative control (cell lysis buffer) confirmed antibody efficacy. Twenty-four organisms were measured for each group (uninjected, morphant-control, and morphant-treatment). Experiments were repeated in triplicate.

Following knockdown of Hsp90ab1, cells were serum-starved for 3 h in serum-free basal media (Promocell, Germany) containing 0.2% BSA prior to stimulation with insulin for 5 min, washed with cold PBS, then lysed with cell lysis buffer provided in the Instant One ELISA kit (eBioscience). Protein concentration was determined using a BCA assay (Thermo Fisher). ELISA for p-Akt and Akt was performed using Instant One ELISA kit. Results are expressed as p-Akt:Akt ratio for each condition.

For this protocol, a commercial kit containing monoclonal antibodies specific to phosphorylated mouse p65 protein (phospho-p65 NF-κB (Ser536) Instant One ELISA kit, eBioscience, San Diego, California, USA) were used. The experimental protocol was performed according to the manufacturer's instructions. Colorimetric measurements (450 nm) were performed on an ELISA plate reader, and results are expressed as relative fold change compared with the negative control group, which represent the basal expression of phosphorylated p65 NF-κB.