Anti misp

Cell lysates were prepared using RIPA buffer (SIGMA; R0278) supplemented with protease inhibitors (Roche; 04693124001). Samples were centrifuged at 20,000 x g for 15 minutes to remove cell debris. The resulting supernatant was boiled with Laemmli sample buffer for 5 minutes. Samples were then loaded on a 4-12% NuPAGE gradient gel (Invitrogen; NP0322BOX). Gels were transferred onto a nitrocellulose membrane at 30V for 18 hours. Membranes were blocked with 5% dry milk diluted in 1X PBS containing 0.1% Tween-20 (PBS-T) for 2 hours at room temperature. The membranes were incubated with primary antibody diluted in 1X PBS-T containing 1% BSA overnight at 4C. Primary antibodies used were anti-MISP (Thermo Scientific; PA5-61995), anti-villin (Santa Cruz; sc-66022), anti-GAPDH (Cell Signaling; 2118), anti-β-actin (SIGMA; A5316). Membranes were then washed with 1X PBS-T and incubated with secondary antibodies for 1 hour at room temperature. Secondary antibodies used were IRdye 800 donkey anti-rabbit (LI-COR; 926-32213) or donkey anti-mouse (LI-COR; 926-32212). Membranes were washed with 1X PBS-T and imaged using the Odyssey CLx infrared scanner (LI-COR). Images were processed using the FIJI software (NIH). Protein expression levels were normalized to GAPDH.

Cells grown on a glass coverlips were fixed with 4% paraformaldehyde (EMS; 15710) in 1X PBS for 15 minutes at 37 °C. Fixed cells were washed with 1X PBS, and permeabilized with 0.1% Triton X-100 in 1X PBS for 15 minutes at room temperature. Cells were washed with 1X PBS and blocked with 5% Bovine Serum Albumin (BSA) in 1X PBS for 2 hours at room temperature. Cells were washed and incubated with primary antibodies overnight at 4 °C. Primary antibodies used were anti-MISP (Thermo Scientific; PA5-61995), anti-villin (Santa Cruz; sc-66022), anti-ezrin (CST; 3145), anti-EPS8 (SIGMA; HPA003897). Cells were washed with 1X PBS four times for 5 minutes and incubate with secondary antibodies. Goat anti-rabbit Alexa Fluor 488 F(ab')2 Fragment (Molecular Probes; A11070), goat anti-mouse Alexa Fluor 568 F(ab')2 Fragment (Molecular Probes; A11070), Alexa Fluor 568-phalloidin (Invitrogen; A12380), Wheat Germ Agglutinin 405M (WGA) (Biotium; 29028-1), DRAQ5 (Thermo Scientific; 62251). Cells were washed again with 1X PBS and mounted on glass slides using ProLong Gold (Invitrogen; P36930).