Secondary goat anti rabbit igg hrp

Cell-permeable inhibitors were purchased as follows: trifluoperazine and W7 calmodulin antagonists from Sigma (Poole, UK); the myristoylated PKA inhibitor Myr-PKI 14-22 amide from Merck Chemicals (Nottingham, UK); and Myr-PKC 19-27 and hypericin from Fisher Scientific (Loughborough, UK). FluoroDish^TM dishes were from World Precision Instruments Ltd. (Stevenage, UK). Monoclonal rabbit anti-AQP4 antibody was from Abcam (Cambridge, UK, product code ab128906), and secondary goat anti-rabbit IgG-HRP was from Santa Cruz Biotechnology. Gateway vectors and enzymes were from Invitrogen (Paisley, UK). Unless otherwise specified, all other chemicals were from Sigma or Fisher Scientific. Cell culture reagents, including calcium-free DMEM, were from Sigma. In each experiment, all inhibitors were analyzed using uninhibited cells transfected with wild-type AQP4-GFP as a positive control.

Cry1Ab oligomers were produced by incubation of 1 μg of purified toxin with 10 μg of BBMV protein in 1 M of buffer NaHCO₃ with a pH of 10.5 during 1 h at 30 °C. After incubation of Cry1Ab toxin with BBMVs, samples were ultra-centrifuged for 30 min at 55,000 rpm, 4 °C and the membrane pellet was recovered for further analysis. Oligomerization reactions were performed in presence or absence of reducing agents (0.02% 2-ME or 5 mM DTT). The samples were suspended in Laemmli loading buffer without 2-ME, heated at 50 °C for 3 min, separated by SDS-PAGE and detected by western blot using polyclonal anti-Cry1Ab and secondary goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA). Finally, oligomers were visualized with Luminol reagent (Santa Cruz Biotechnology, Dallas, TX, USA) in Amersham Imager 600 device (GE LifeScience, Little Chalfont, UK).