Hrp labeled goat anti mouse rabbit secondary antibody

Cells were homogenized briefly in 10 volumes of lysis buffer containing (in mM) 20 Tris–HCl (pH 7.4), 150 NaCl, 2.5 EDTA, 50 NaF, 0.1 Na₄P₂O₇, 1 Na₃VO₄, 1 PMSF, 1 DTT, 0.02% (v/v) protease cocktail (Sigma Aldrich, St. Louis, MO, USA), 1% (v/v) Triton X-100 and 10% (v/v) glycerol. The homogenates were centrifuged twice at 20,000×g at 4 °C for 15 min, and the supernatants were retained as total protein. Protein concentrations were determined by the BCA method. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, MA, USA). Western blot analysis was performed under standard conditions with specific anti-B7-H6 (1:2000; Abcam, MA, USA), anti-C-myc (1:1500, Abcam, MA, USA), anti-C-fos (1:2000, Cell Signaling Technology, MA, USA), anti-cyclin D1 (1:2000, Cell Signaling Technology, MA, USA), and anti-GAPDH (1:4000, Sigma, St. Louis, MO, USA) antibodies and HRP-labeled goat anti-mouse/rabbit secondary antibody (1:6000, Sigma Aldrich, St. Louis, MO, USA). The immunoreaction was visualized using an enhanced chemiluminescence detection kit (Thermo Fisher, MA, USA) and exposure to X-ray film, and band densities were quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad).

The Western blotting analysis was performed according to the protocol we have reported [28 (link)–30 (link)]. The anti-HHLA2 (1:2000; Abcam, MA, USA), the anti-GAPDH (1:4000, Sigma, St. Louis, MO, USA), and the HRP-labeled goat anti-mouse/rabbit secondary antibody (1:6000, Sigma Aldrich, St. Louis, MO, USA) were used, and the immunoreaction was visualized using an enhanced chemiluminescence detection kit (Thermo Fisher, MA, USA) and exposed to X-ray film, and band densities were quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad).

The Western blotting analysis was performed as previously described [6 (link)]. The antibodies against METTL3 (1:2,000; Catalog No. ab195352, Abcam, MA, USA), HHLA2 (1:2,000; Catalog No. ab214327, Abcam, MA, USA), and GAPDH (1:4,000, Sigma, St. Louis, MO, USA) were used in the present study, and the HRP-labeled goat anti-mouse/rabbit secondary antibody (1:6,000) was purchased from Sigma Aldrich (St. Louis, MO, USA). The immunoreactive bands were examined by an enhanced chemiluminescence detection kit (Thermo Fisher, MA, USA), and then exposed to X-ray film, and band densities were quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad).