Hrp conjugated goat anti rabbit igg secondary antibody

Cellular protein was extracted and analyzed as previously described [16 (link), 17 (link)]. Briefly, cells were washed three times with ice-cold PBS and lysed in lysis buffer containing 50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0.1% NP40, 20% glycerol, and protease inhibitor cocktail (Complete Mini, Roche, Indianapolis, IN) for 1 h. Cells were then transferred to a 1.5 ml tube and centrifuged. The supernatant was transferred into a new tube, and then the protein concentration was measured. Proteins (10 μg) were heated in SDS sample loading buffer at 70°C for 10 min and loaded onto NuPage 4–12% Bis-Tris gels (Thermo Fisher Scientific). After electrophoresis, proteins were transferred to a PVDF membrane using an iBlot 2 transfer system (Thermo Fisher Scientific). The membrane was washed with PBST (PBS with 0.1% Tween 20), blocked overnight with ImmunoBlock (KAC Co., Ltd., Kyoto, Japan), and then incubated with anti-GPAT3 (Thermo Fisher Scientific; 1:2,000 dilution) or anti-β-actin (Cell Signaling Technology, Danvers, MA; 1:2,000 dilution) antibodies. After washing with PBST, the membrane was incubated for 1 h with HRP- conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific; 1:1,500 dilution). The signal was developed using Amersham ECL Prime (GE Healthcare, Buckinghamshire, UK), and the images were scanned with an Amersham Imager 680 RGB (GE Healthcare).

Proteins from LSCs were extracted with a cold lysis buffer composed of protease and phosphatase inhibitors (ThermoFisher Scientific, Waltham, MA, USA). Protein concentration was measured by a BAC protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). Equal amounts of protein extracts (15 μg) were subjected to electrophoresis on 10% acrylamide gels and then transferred to polyvinylidene difluoride membranes (Roche, Basel, Switzerland). After being blocked in 5% BSA for 1 h at 25 °C, the membranes were incubated overnight at 4 °C with primary antibodies for anti-p63 (1:1000 dilution, ab124762, Abcam, Cambridge, UK), C/EBPδ (1:1000 dilution, ab65081, Abcam, Cambridge, UK), and K12 (ab185627, Abcam, Cambridge, UK). After 3 washes with Tris-buffered saline containing 0.05% Tween-20 for 10 min each, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1: 5000 dilution, 31460, ThermoFisher Scientific, Waltham, MA, USA) for 1 h at room temperature. An HRP-conjugated mouse anti-β-actin antibody (1:1000 dilution, A3854, Sigma-Aldrich, Saint Louis, MO, USA) was used for protein quantification. The results were detected by an enhanced chemiluminescence reagent kit (NCM Biotech, Suzhou, China) and recorded by the transilluminator (Bio-Rad, Hercules, CA, USA).

Equal protein aliquots of BALFs were resolved by SDS-PAGE. In order to adequately visualize the > 250 kDa MUC1-ED protein, discontinuous gel electrophoresis was performed under reducing conditions in 3% stacking/5% separating acrylamide gels containing 25 mM Tris–HCl, 190 mM glycine, 0.1% SDS, pH 8.3 until the 150 kDa prestained protein molecular weight marker reached the bottom of the gel. The resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA) and the membranes probed with rabbit anti-mouse MUC1-ED antibody, followed by HRP-conjugated goat anti-rabbit IgG secondary antibody and enhanced chemiluminescence reagents (Thermo Fisher Scientific), as described^{22 (link)}.