Horseradish peroxidase conjugated anti mouse or rabbit antibody

Cells were lysed in SDS-sample buffer [62.5 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, and 1% (w/v) SDS] with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 2 μg ml⁻¹ pepstatin, 2 μg ml⁻¹ leupeptin, and 2 μg ml⁻¹ aprotinin) and phosphatase inhibitors (5 mM NaF and 2 mM sodium orthovanadate). After sonication, the lysates were used as the total protein extracts. The protein concentrations of samples were determined by the BCA assay (Pierce). Proteins were recovered with an equal volume of 2× SDS-sample buffer [125 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 200 mM dithiothreitol (DTT) and 0.01% (w/v) bromophenol blue]. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% (w/v) BSA in TTBS buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% (w/v) Tween 20] and incubated with an antibody to GAPDH (6C5; Calbiochem), monoclonal mouse anti-LPIAT1 antibody (clone: FT10)^{23 (link)}, VPS34 (sc-365404; Santa Cruz), Pikfyve (sc-100408; Santa Cruz), PI4Kα (12411-1-AP; Proteintech), and PI4Kβ (611816; BD laboratory). After incubation with horseradish peroxidase-conjugated anti-mouse or -rabbit antibody (GE Healthcare), the protein was detected by enhanced chemiluminescence (ECL Western blotting detection system, GE Healthcare).

Whole cell lysates were extracted using either NETN buffer supplemented with protease inhibitor cocktail or direct lysis. Protein lysate was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (Millipore) for western blot analyses. Primary antibodies against CDK6, CDK4 [EPR2513Y], MAP3K7 (TAK1) [EPR5984] (1:1000, ab151247, ab68266, ab109526, Abcam), p-GSK3β (Ser9) (D3A4), β-catenin (D10A8), p44/42 MAPK, p-p44/42 MAPK, YAP (D8H1X), pYAP(Ser127) (D9W2I), SOX2 (D6D9), RPS6KA4 (MSK2) (D41A4) (1:1000, #9322 S, #8480 S, #9102 S, #9101 S, #14074 S, #13008, #3579, #3679, Cell Signalling Technology), GSK3β (clone 7) (1:1000, #610202, clone 7, BD Transduction Laboratories), HA (1C1D2) (1:1000, #66061-1-Ig-AP, Proteintech), OCT4 (C-10) (1:1000, sc5279, Santa Cruz) and β-ACTIN (AC-74) (1:5000, #A5316, Sigma Aldrich) were incubated at 4 °C overnight. After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse or rabbit antibody (GE HealthCare). The signals were visualized using the enhanced chemiluminescence method. Blot images were quantified by densitometry using ImageJ software.