Hrp conjugated β actin antibody

Reagents were purchased from Sigma-Aldrich (e.g., S1P, thrombin) except as noted below. Dulbecco’s phosphate buffered saline (PBS) and trypsin were obtained from Life Technologies, while biological grade glutaraldehyde and paraformaldehyde were obtained from Electron Microscopy Science. The following reagents and antibodies were acquired for immunofluorescence: mouse monoclonal anti-cortactin (p80/85) antibody clone 4F11 (EMD Millipore), mouse anti-paxillin antibody clone 349 (BD Biosciences), mouse anti-VE-cadherin antibody (Santa Cruz), Alex Fluor 488 goat anti-mouse IgG secondary antibody (Life Technologies), HRP-conjugated β-actin antibody (Proteintech Group Inc), and Rhodamine-conjugated phalloidin (Molecular Probes). Scrambled and c-Abl specific siRNA was obtained from Santa Cruz Biotechnology, and Xfect RNA transfection reagent was obtained from Takara Clontech.

Western blotting analysis was carried out in accordance with the procedures outlined in the previous section (Mo et al., 2020 (link)). SDS-PAGE and polyvinylidene fluoride membranes were used to separate the protein lysates. The membranes were blocked for 1 h in Tris-buffered saline containing 0.1% Tween 20 before being probed with specific antibodies. Primary antibodies included p-AKT^Thr308, t-AKT, p-mTOR, p-RPS6, SREBP1, FASN, ACC, HK2, and PKM2. Anti-rabbit/mouse IgG/HRP (secondary antibody) was added following incubation with each primary antibody. HRP-conjugated β-actin antibody from Proteintech (Wuhan, China) was used as an internal standard. Using SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical, Ridgewood, New York, United States) and a chemiluminescence imager, immunoblotting was performed and photographed (G: BOX Chemi XRQ; Syngene, Cambridge, United Kingdom).
The IHC assay was performed as previously described (Hu et al., 2016 (link)). Specific primary antibodies used in immunohistochemical staining are listed in Supplementary Table S1.

Proteins in the mouse liver specimens were extracted using Mammalian Protein Extraction Reagent (Thermo Scientific, Waltham, MA) containing Cocktail Protease Inhibitor (Roche, Indianapolis, IN, USA). A BCA protein determination assay was performed for quantification of the protein concentration. The lysates were denatured at 95 °C in Tris–glycine SDS loading buffer. After the gradient was separated using SDS-PAGE and blotted to polyvinylidene fluoride membranes, the membranes were blocked, and the proteins on the membranes were probed with specific primary antibodies. The following primary antibodies (CST, USA) were used: phosphor-AKT (Thr308), phosphor-AKT (Ser473), total AKT (t-AKT), phospho-S6 ribosomal protein (Ser235/236), PKM2 (D78A4), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), and total ERK1/2 (t-ERK1/2). Each protein linked with primary antibody was further incubated with horseradish peroxidase-secondary antibody for 1 hour at 25 °C. The protein expressions were exhibited with enhanced chemiluminescence (ECL) reagent (Thermo Scientific, USA). The HRP-conjugated β-actin antibody (Proteintech, Wuhan, China) served as an internal standard for normalization.