Total RNA was isolated from cells using the Steady Pure Quick RNA extraction kit (Accurate Biotechnology, AG21023). The isolated RNA was reverse-transcribed into complementary DNA using the Evo Moloney-murine leukemia virus reverse transcription premix kit (Accurate Biotechnology, AG11728). qRT-PCR was performed in a LightCycler (Roche, Germany) with 2× Universal SYBR Green Fast qPCR Mix (Abclonal, RK21203). The mRNA expression of LPCAT1 was calculated using the 2−ΔΔCt method. The primer sequences are shown in Table S2. Western blotting was performed a western blot as described previously (26 (link)). The following antibodies were used in western blotting analysis: anti-LPCAT1 antibody (1:500, Proteintech, 16112-1-AP), anti-CASP1 (1:6000, Proteintech, 22915-1-AP), anti-CASP3 (1:500, ABclonal, A2156), anti-CASP7 (1:600, Proteintech, 27155-1-AP), anti-CASP8 (1:500, ABclonal, A0215), anti-GSDME (1:5000, Proteintech, 13075-1-AP), anti-cleaved GSDME antibody(1:500, ABclonal, A23072), anti-GSDMD (Full Length+N terminal, 1:500, ABclonal, A20197), anti-MLKL (1:500, ABclonal, A21894), anti-phospho-MLKL antibody(1:50, ABclonal, A1244), and anti-GAPDH antibody (1:10000, Proteintech, 60004-1-Ig).
Free full text: Click here