Anti myosin iia antibody

Cells were fixed with 3.7% formaldehyde (Sigma) in PBS for 10 min at RT, followed by PBS washing 2× for 5 min. For microtubule staining, cells were fixed with 2.5% formaldehyde and 0.15% glutaraldehyde in PBS for 10 min at RT followed by quenching with 0.5 mg/ml sodium borohydride (Sigma) in PBS for 5 min 3×. Blocking and antibody staining was done in 1% BSA in PBS. Rabbit polyclonal ant-pericentrin antibody (Abcam) was used to stain for the MTOC at 1:1000 dilution, anti-myosin IIA antibody (Sigma) was used at 1:100, anti-myosin IIB antibody (Cell Signaling) was used at 1:150, anti-α-tubulin (Sigma) at 1:2000 and all primary antibodies were incubated at RT for 1 hour or overnight at 4°C. All secondary antibodies (Alexa dyes 488, 564, 647) were used in staining for 1 hour at RT at 1:300 dilution. TRITC-phalloidin (Sigma) was used with the donkey secondary antibodies at a concentration of 100 ng/ml and Hoechst 33342 (Invitrogen) was used to stain DNA at a concentration of 1 µg/ml for 10 min at RT. Imaging for quantification of MII levels and localization was performed using an inverted microscope (IX-71, Olympus) with a 40× LUCPlanFLN objective (NA 0.60) and a Cascade CCD camera (Photometrics). Image acquisition was performed with Image Pro software (Media Cybernetics, Inc.) and subsequently background was subtracted and image analysis was done using ImageJ.