Takara bca protein assay kit

Cells were collected, washed once with cold PBS, and lysed at 4 °C for 15 min in lysis buffer [1% NP40, 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktails (Nacalai Tesque, Kyoto, Japan), 1 mM Na₃VO₄, and 50 mM NaF], they were then centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatant was collected, and the amount of protein in the cell lysate was measured using a TaKaRa BCA Protein Assay Kit (TaKaRa Bio, Shiga, Japan) according to the manufacturer’s recommendations. The samples were separated on a 10% acrylamide gel before proteins were blotted onto a PVDF membrane (Merck, Darmstadt, Germany). After blotting, the membrane was blocked with blocking buffer (Tris-buffered saline with 0.05% Tween 20 and 5% skim milk) for 1 h at room temperature. The membrane was incubated with a primary antibody, mouse monoclonal anti-FLAG antibody (M2; Sigma-Aldrich; dilution 1:1000), an anti-fPD1 antibody in TBS-T with 0.5% skim milk at 4 °C overnight. The membrane was washed 3 times with TBS-T for 10 min at a time and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Biolegend Japan, Tokyo, Japan; dilution 1:5000) for 1 h. The membrane was imaged with an Amersham ImageQuant 800 after being washed a further three times with TBS-T for 10 min (Cytiva).

After treatment of cells with siRNAs for 3 days without exchanging medium, cells were rinsed twice with ice-cold D-PBS and subsequently lysed in radio-immunoprecipitation assay (RIPA) buffer, containing protease inhibitor cocktails, for 30 min on ice. The cell debris were removed by centrifugation (15,000× g, 4 °C for 10 min) and the supernatant of the resulting suspension was collected as the total cell lysate. Protein concentration was quantified using the TaKaRa BCA Protein Assay Kit (Takara Bio).

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and Escherichia coli (E. coli) extract polar were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). PUREfrex2.0 was purchased from Cosmo Bio (Tokyo, Japan). A pre-cast gel of 15% SDS-polyacryamide gel was purchased from ATTO (Tokyo, Japan). HEPES was purchased from DOJINDO LABORATORIES (Kumamoto, Japan). KCl and sucrose were purchased from FUJIFILM Wako Chemicals (Tokyo, Japan). TaKaRa BCA protein assay kit was purchased from TaKaRa BIO (Shiga, Japan).