Goat anti rabbit igg alexafluor 680

RBC membranes were obtained by lysing one volume of packed RBCs in 10 volumes of ice-cold lysis buffer (5 mM Na₂HPO₄, pH 8.0 with protease inhibitors (cocktail tablet; Roche, Basel, Switzerland), 3 mM benzamidine and 1 mM Na₃VO₄) for 10 min on ice. The membrane fraction was washed repeatedly by centrifugation at 21,000xg for 10 min to remove free hemoglobin and solubilized 1:1 with Laemmli buffer (Bio-Rad Laboratories, Hercules CA, USA) supplemented with 100 mM β-mercaptoethanol for SDS-PAGE. SDS-PAGE was carried out according to Laemmli [11 (link)] on 10% polyacrylamide gels. Proteins were transferred to PVDF membranes using the iBlot system (Invitrogen, Carlsbad CA, USA). These membranes were blocked with Odyssey Blocking Buffer (LI-COR, Lincoln NE, USA) and probed with 1:2000 mouse anti-band 3 N-terminal domain (BIII-136, Sigma-Aldrich, St. Louis MO, USA), 1:5000 mouse anti-β-spectrin (Acris, Herford, Germany), or 1:10000 anti-β-actin (Sigma-Aldrich). As secondary antibodies 1:10000 goat anti-rabbit IgG-Alexa Fluor 680 (Invitrogen, Carlsbad CA, USA), and/or goat anti-mouse IgG-IRDye 800 (LI-COR, Lincoln NE, USA) were used. The blots were scanned with the Odyssey Infrared Imaging System (LI-COR, Lincoln NE, USA) and the images were analyzed using the Odyssey Software version 2.1.

MEFs (50,000/well) were plated in 96-well plates and serum-starved overnight before stimulation with 10% FBS for 5 min ±10 µM Y27632. Staining for pMLC was performed as previously described (Unbekandt et al., 2014 (link)), using rabbit anti-pMLC2 Thr18/Ser19 (#3674; Cell Signaling Technology), and detected with goat anti-rabbit IgG Alexa Fluor 680 (A12076; Invitrogen). Total cell number was determined by nuclei staining with the fluorescent marker DRAQ5 (1:700, Biostatus). Fluorescence was determined by imaging the plate with an Odyssey LiCOR scanner and signal intensity with Odyssey software. pMLC signal was normalized to DRAQ5 signal.