Mouse monoclonal igg2b

The experimental details provided below for Western blotting experiments complies with BJP’s Guidelines for immunoblotting and immunohistochemistry (Alexander et al., 2018 (link)). HEK293 cells with endogenous β₂AR were stimulated with 10 μM β-agonist for 0, 2, 5, 10, 15, 20, 25, or 30 minutes. The cells were lysed in Laemmli buffer, and equal amounts of protein were analyzed by SDS PAGE and Western blot. The membranes were incubated overnight at 4 °C with anti-phospho-ERK 1/2 (P-p44/42 MAPK; rabbit mAB; Ref:4370S; Lot 31; 1:5000; Cell Signaling Technologies, Danvers, MA, USA) and anti-total ERK (ERK1/2 (C-9); sc-514302; Lot #I19.3; mouse monoclonal IgG_2B; 1:10000; Santa Cruz Biotechnology, Dallas, TX, USA) followed by IRDye conjugates (IRDye800CW, goat anti-Mouse, 926–32210, Lot C91210–09/ IRDye680RD, goat-anti-rabbit 926–68071, Lot D00115–06; 1:300; Li-Cor Biosciences, Lincoln, NE, USA) as secondary antibodies. Protein bands were detected with a LI-Cor Odyssey CLX far-infrared scanner (Li-Cor Biosciences, Lincoln, NE, USA) and the bands were quantified by densitometry with ImageJ v. 1.53 (RRID:SCR_003070) (Schneider et al., 2012 (link)).