A600020

PIK3C3/VPS34 kinase activity assay was performed as described previously [11 ]. Briefly, PIK3C3 was immunoprecipitated using an anti-ATG14 antibody after PVT1 or miR-619-5p overexpression with or without gemcitabine treatment. Then the immunoprecipitates were incubated with kinase reaction buffer (100 mM HEPES [ThermoFisher, 15,630,080] [pH 7.5], 300 mM NaCl, 2 mM CHAPS [ThermoFisher, 28,300], 10 mM MnCl2, 2 mM DTT, 100 μM ATP [Sangon Biotech, A600020]) and phosphatidylinositol substrate (Echelon, K-3000) and incubated at room temperature for 2 h. After the addition of the PtdIns3P detection buffer (provided by the kit, K-3004) and PtdIns3P detector protein (provided by the kit), the reaction mixture was then added to the PtdIns3P-coated microplate. The amount of PtdIns3P detector protein bound to the plate was determined through colorimetric detection of the absorbance at 450 nm. The concentration of PtdIns3P in the reaction mixture was calculated as the inverse of to the amount of PtdIns3P detector protein bound to the plate.

PIK3C3/VPS34 kinase activity assay was performed as described previously (Zhou et al., 2020a (link)). Briefly, PIK3C3 was immunoprecipitated using an anti-ATG14 antibody after co-transfection with EGFR L858R/Δ746-750 mutant and MYC-STYK1 in the condition of starvation or gefitinib treatment. Then the immunoprecipitates were incubated with kinase reaction buffer (100 mM HEPES [ThermoFisher, 15,630,080] [pH 7.5], 300 mM NaCl, 2 mM CHAPS [ThermoFisher, 28,300], 10 mM MnCl2, 2 mM DTT, 100 μM ATP [Sangon Biotech, A600020]) and phosphatidylinositol substrate (Echelon, K-3000) and incubated at room temperature for 2 h. The concentration of PtdIns3P in the reaction mixture was calculated according to the manufacturer's instructions (Echelon, K-3000).