Rabbit anti β actin

Cell extracts were lysed in SDS buffer, a protease inhibitor cocktail (Promega) and a phosphatase inhibitor cocktail (FUJIFILM Wako Pure Chemical Corporation). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS‐PAGE (polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk and probed with the following primary antibodies: goat anti‐galecint‐1 (1:1000, R&D systems), mouse anti‐TSC22D3 (1:500), mouse anti‐ubiquitin (1:500, Santa Cruz Biotechnology), rabbit anti‐HIF‐1α (1:1000), rabbit anti‐phosphorylated AKT (1:2000), rabbit anti‐AKT (1:2000), rabbit anti‐phosphorylated ERK1/2 (1:2000), rabbit anti‐ERK1/2 (1:2000, Cell signaling technology) and rabbit anti‐β‐actin (1:4000, Medical & Biological Laboratories) antibodies. Horseradish peroxidase‐conjugated anti‐goat, anti‐mouse and anti‐rabbit IgGs (Jackson ImmunoResearch Laboratories) were used as a secondary antibody for chemoluminescence detection. Signals were visualized using a SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).

Collected cells were lysed in SDS buffer and sonicated on ice. After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific, Waltham, MA, USA), proteins were resolved by SDS-PAGE and transferred to PVDF membrane by electroblot analysis. Membranes were blocked in Tris-buffered saline containing 5% skim milk and 0.05% Tween-20 and incubated with the following primary antibodies: goat anti-VEGFR3 (1:1000) (AF349) (R&D systems) and rabbit anti-β actin (1:8000) (Medical and Biological Laboratories, Nagoya, Aichi, Japan). Horseradish peroxidase conjugated anti-goat, anti-rabbit IgG (1:4000) were used as secondary antibodies for chemiluminescence detection. Signals were obtained by enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA). The bands were analyzed by densitometry using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).