Fig. 1

SH3BGRL3 colocalizes with both ErbB2 and Myo1c but interacts solely with Myo1c. (a) Upper: representative confocal image of SKBR3 cells transfected with FLAG-SH3BGRL3 and stained with anti-FLAG and anti-ErbB2 mAbs. . Scale bar = 10 μm. Lower: co-localization levels expressed as % of SH3BGRL3-positive pixels that are also positive (over threshold) for ErbB2 fluorescence, evaluated either on the whole FLAG-SH3BGRL3-transfected cell (left) or on ROIs gating the plasma membrane region (right) (see Methods). (b) WB analysis with anti-Myo1c antibody of Co-IP (upper inset) and lysates (middle inset) from transfected cells (left lane) or from cells transfected with an empty vector (right lane). The lower inset displays the lysates revealed with an anti-FLAG antibody to verify the presence of the FLAG-SH3BGRL3 bait. (c) Co-IP of endogenous Myo1c and ErbB2 using FLAG-SH3BGRL3 (left lane), and the empty vector as negative control (right lane). WB analysis with anti-Myo1c antibody of Co-IP and lysates (two upper insets), WB analysis with anti-ErbB2 antibody of Co-IP and lysates (third and forth insets). The lower inset displays the lysates revealed with an anti-FLAG antibody to verify the presence of the FLAG-SH3BGRL3 bait. (d) Upper: representative confocal image of SKBR3 cells transfected with FLAG-SH3BGRL3 and stained with anti-FLAG, anti-Myo1c and anti-ErbB2 antibodies. Arrows point to structures with a morphology that highly resembles membrane ruffles. Scale bars = 10 μm. Lower: co-localization levels expressed as % SH3BGRL3-positive pixels that are also positive (over threshold) for Myo1c or ErbB2 fluorescence, evaluated either on the whole FLAG-SH3BGRL3-transfected cell (first two bars from the left) or on ROIs gating the plasma membrane region (two subsequent bars) (see Methods). To further evaluate whether the SH3BGRL3/ Myo1c co-localizing spots on plasma membranes were SH3BGRL3/ ErbB2 positive as well, the triple SH3BGRL3/Myo1c/ErbB2 co-localization levels were addressed by measuring the % of Myo1c/ErbB2 co-localizing pixels that also contained SH3BGRL3-positive fluorescence (last bar from left) (see Methods)