Classic ip kit

Viruses infected K562 cells were incubated with AP21967 for 24 h and were then collected and lysed with M-PER mammalian protein extraction reagent supplemented with proteinase and phosphatase inhibitors. Co-immunoprecipitation was performed according to the manual of the ProFound^TM Mammalian HA-Tag IP/Co-IP Kit and Classic IP Kit (Thermo Scientific, Walham, MA, USA). Immunoblot analysis was the same as the above description.

INS-1 cells were plated into 6-well plates and then incubated in 5% CO₂ for 48 h for Co-IP assay incubated with normal cell culture. IP was performed by the Classic IP kit (ThermoFisher, CA, USA). The cell (1 mg of protein) was lysed with lyse buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, pH 7.0) and was used for IP with anti-TXNIP or anti- PKA Cα antibodies at 4°C for 12 h. Proteins were then centrifuged and pellets were washed 3 times with cold TNTE 0.1% (50 mM Tris–HCl buffer pH 7.4, 150 mM NaCl, 5 mM EDTA and 0.1% Triton X-100). Pellets were re-suspended in Laemmli buffer (100 mM Tris–HCl pH6.8, 20% glycerol, 2% SDS, 0.05% bromophenol blue and 100 mM DTT) and boiled for 5 min. The proteins were analyzed by western blotting assay.

Immunoprecipitation assays were performed using a Classic IP Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. NSC34 cells were plated at 1.4 × 10⁵cells/well in 6-well plates (BD Biosciences) and incubated overnight. The cell lysates were incubated with mouse anti-NKA α1 antibody, mouse anti-NKA α3 antibody, or normal mouse IgG (Santa Cruz Biotechnology). A mixture of equal parts of a protein sample and sample buffer with 20% 2-mercaptoethanol (Wako, Osaka, Japan) was subjected to SDS-PAGE (Wako). The separated proteins were then transferred onto a polyvinylidene difluoride membrane (PVDF, Immobilon-P; Merck KGaA, Darmstadt, Germany). To visualize the proteins on the membrane, the primary antibody was goat anti-GPNMB antibody (1: 500, R&D Systems Inc., Minneapolis, MN, USA) and the secondary antibody was horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (1: 2000, Thermo Fisher Scientific). The immunoreactive bands were visualized using a chemiluminescent substrate (ImmunoStar^® LD; Wako). The band intensity was measured using an imaging analyzer (LAS-4000; Fuji Film, Tokyo, Japan).