Hrp conjugated anti mouse or anti rabbit secondary abs

Twenty micrograms of total protein lysates were separated on an SDS polyacrylamide gel, transferred onto Hybond-C extra membranes (GE Healthcare, Milan, Italy), blocked with 5% low-fat dry milk in PBS-Tween 20, immunoblotted with goat anti-CD274 (PD-L1, 0.5 μg/ml, R&D System, Minneapolis, MN, United States), mouse anti-CD273 (PD-L2, 1 μg/ml, R&D System), rabbit anti-pAKT (1:1.000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-AKT (1:1.000, Cell Signaling), mouse anti-pERK (1:2.000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-ERK (1:1.000, Cell Signaling Technology), and anti-glyceraldehydes-3-phosphate dehydrogenase (GAPDH, 1:8.000, OriGene) antibodies (Abs) for 1 h and then incubated with HRP-conjugated anti-mouse or anti-rabbit secondary Abs (1:2.000, Cell Signaling Technology) and with HRP-conjugated anti-goat secondary Ab (1:1.000, Cell Signaling Technology) for 1 h. Peroxidase activity was visualized with the LiteAblot^®PLUS or TURBO (EuroClone, Milan, Italy) kit, and densitometric analysis was carried out by a Chemidoc using the Quantity One software (Bio-Rad).

20 ug of the lysates were separated on a sodium dodecyl sulfate (SDS) polyacrylamide gel, transferred onto Hybond-C extra membranes (GE Healthcare, Milan, Italy) and blocked with 5% low-fat dry milk in PBS-Tween 20 (Sigma-Aldrich). Immunoblots were incubated with goat polyclonal anti-TRPV2 (1:300, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-LC3 (2 μg/mL, Novus Biologicals, Centennial, CO, USA), rabbit anti-pAKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-AKT (1:1000, Cell Signaling Technology) and rabbit anti-glyceraldehydes-3-phosphate dehydrogenase (GAPDH, 1:8000, OriGene Technologies, Rockville, MD, USA) antibodies (Abs) for 1 h and then with HRP-conjugated anti-mouse or anti-rabbit secondary Abs (1:2000, Cell Signaling Technology, Danvers, MA, USA) and with HRP-conjugated anti-goat secondary Ab (1:1000, Cell Signaling Technology, Danvers, MA, USA) for 1 h. Peroxidase activity was visualized with the LiteAblot^®PLUS or TURBO (EuroClone, Milan, Italy) kit and densitometric analysis was carried out by a Chemidoc using the Quantity One software (Bio-Rad, Milan, Italy).