Cf633 phalloidin

To investigate localization of Mlph mutants, immunocytochemistry was performed in a quail myoblast cell line (QM7). Vectors containing each of the Mlph mutants were co-transfected with CD9 or FYVE vectors. After 48 h of the incubation, cells were washed, and subsequently permeabilized after fixation with 10% neutral buffered formalin for 1 h. Anti-Flag antibodies were used for detecting Mlph mutants, and Alexa fluor 488 conjugated anti-mouse IgG (#A11011, Invitrogen, Waltham, MA, USA) was used as a secondary antibody. In addition, Phalloidin-CF633 (#00046, Biotium, Hayward, CA, USA) and Lysoview (#70058, Biotium) were used for staining F-actin and lysosome, respectively. There was use of DAPI for nuclei counterstaining.

Actin (12.5 µM) in G-buffer was mixed with 2 mM MgCl₂, 5 mM EGTA and 100 mM KCl for 1 h at 24°C. To make a 0.5 mM Latrunculin A (LatA) solution, 10 mM LatA in DMSO was first diluted to 2 mM with G-buffer, then further diluted to 0.5 mM in the reaction mixture (the final concentration of DMSO was 4%). The reaction mixture was ultracentrifuged in an Airfuge (Beckman Coulter) at 25 psi, room temperature for 30 min. Pellet and supernatant fractions were analysed on Coomassie-stained gels. For imaging actin filaments, actin (8.33 µM) in G-buffer was polymerized by addition of 2 mM MgCl₂, 5 mM EGTA, 100 mM KCl and 2% polyethylene glycol 20,000 (#8.17018.1000, Merck Millipore) followed by incubation at 24°C for 1 h. 5% (v/v) Rhodamine–phalloidin (#R415, Thermo Fisher Scientific) or phalloidin–CF633 (#00046, Biotium) was added, and F-actin was imaged using an Andor Revolution XD spinning-disc confocal system on a Nikon Eclipse Ti inverted microscope, with a Nikon Plan Apo Lambda 100×1.45 NA oil immersion objective lens, a spinning-disc system (CSU-X1; Yokogawa) and an Andor iXon Ultra EMCCD camera. Images were acquired at the pixel size of 80 nm/pixel by using the Andor IQ3 software. Laser lines at a wavelength of 561 or 640 nm was used for excitation.