Donkey anti goat igg hrp sc 2033

Cells were plated at 3 million cells/plate and treated with the indicated concentrations of inhibitors for 48 hrs. Cells were lysed in RIPA (0.15 M NaCl, 0.05 M Tris HCl, 1% w/v sodium deoxycholate, 1% w/v SDS, 1% w/v Triton X-100, 1 mM EDTA) or TX-114 buffer containing protease inhibitor cocktail and phenylmethanesulfonyle fluoride. Protein concentrations were quantified by the BCA method.[50 (link)] Equal protein quantities were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane overnight. Membranes were blocked for 45 min in 5% non-fat milk followed by incubation with primary antibodies overnight and secondary antibodies for 1 hr. Protein levels were visualized using ECL detection site (GE Healthcare, Buckinghamshire, UK). Primary antibody anti-pan-Ras was obtained from Inter-Biotechnology (Tokyo, Japan), Rap1A (sc-1482), Rab6 (sc-310), β-tubulin (sc-9140), calnexin (sc-23954), and secondary antibody donkey anti-goat IgG-HRP (sc-2033) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and secondary HRP-conjugated anti-rabbit (NA934) and anti-mouse (NXA931) antibodies were obtained from GE Healthcare (Buckinghamshire, UK).

K562 cells were plated at 3 million cells/plate and immediately treated for 48 hrs. Cells were lysed in RIPA buffer (0.15 M NaC1, 0.05 M Tris HCl, 1% w/v sodium deoxycholate, 1% w/v SDS, 1% w/v Triton X-100, 1 mM EDTA (Research Products International Corp, Mount Prospect, IL)) or TX-114 as indicated, both containing protease inhibitor cocktail and phenylmethanesulfonyl fluoride (Sigma Aldrich, St. Louis, MO). Protein concentrations were quantified by BCA assay [28 (link)]. Equal protein quantities were resolved by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes overnight. Membranes were blocked at 37 °C for 45 min in 5% non-fat milk followed by incubation with primary antibodies at 4 °C overnight and secondary antibodies at 37 °C for 1 hr. Proteins were visualized using ECL detection (GE Healthcare, Buckinghamshire, UK) and quantified by densitometry using Image J. [29 ] Anti-pan-Ras was obtained from Inter-Biotechnology (Tokyo, Japan). Rap1A (sc-1482), Rab6 (sc-310), β-tubulin (sc-9140), calnexin (sc-23954), and secondary antibody donkey anti-goat IgG-HRP (sc-2033) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary HRP-conjugated anti-rabbit (NA934) and anti-mouse (NXA931) antibodies were obtained from GE Healthcare (Buckinghamshire, UK).