Monoclonal gapdh antibody mab374

Cells were treated with M-PER^® Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA), HALT Protease Inhibitor Cocktail (Thermo Scientific), and Phosphatase Inhibitor Cocktail (Thermo Scientific) to extract total protein. Approximately, 15–20 µg of total protein were electrophoresed per well on an 8–10% SDS-polyacrylamide gel and transferred onto Immobilon-FL PVDF membranes (Millipore, Billerica, MA, USA). Blots were incubated with the HIF-1α primary antibody (H1alpha 67): sc-53546 (Santa Cruz Biotechnology, CA, USA) at 1:500 dilution and monoclonal GAPDH antibody (MAB374, from Millipore, Billerica, MA, USA) at 1:10,000 dilutions overnight. After blocking with primary antibodies, membranes were washed and then probed with a mixture of IRDye polyclonal secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Images were read with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Cancer cells were treated with M-PER^® Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA), HALT Protease Inhibitor Cocktail (Thermo Scientific), and Phosphatase Inhibitor Cocktail (Thermo Scientific) to extract total protein. A total of 15–20 µg of total protein were electrophoresed per well on a SDS-polyacrylamide gel; transferred onto Immobilon-FL PVDF membranes (Millipore, Billerica, MA, USA); and further incubated with the following primary antibodies: VEGFA (sc-507), VEGFC (sc-1881), VEGFD (sc-13085), LYVE1 (sc-28190), and COX-2 (sc-1747), using antibodies (1:500 dilutions) from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Monoclonal GAPDH antibody (MAB374) was from Millipore, Billerica, MA, USA. After blocking with primary antibodies, overnight blots were probed with a mixture of IRDye polyclonal secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Images were read with an Odyssey infrared imaging system (LI-COR Biosciences).